Polymers containing polysaccharides such as alginates or modified alginates

ABSTRACT

Materials which contain polysaccharide chains, particularly alginate or modified alginate chains. The polysaccharide chains may be included as side chains or auxiliary chains from a backbone polymer chain, which may also be a polysaccharide. Further, the polysaccharide chains may be cross-linked between side chains, auxiliary chains and/or backbone chains. These materials and non-modified or otherwise modified alginate materials are advantageously modified by covalent bonding thereto of a biologically active molecule for cell adhesion or other cellular interaction. Processes for preparation of these alginate materials and methods for using them, particularly for cell transplantation and tissue engineering applications.

Priority is claimed to the following U.S. provisional applications: Ser.No. 60/026,362 filed Sep. 19, 1996; Ser. No. 60/026,467 filed Sep. 19,1996; and Ser. No. 60/041,565 filed Mar. 21, 1997.

This application is a 371 of PCT/US97/16890, filed Sep. 19, 1997.

The invention relates to materials which contain polysaccharide chains,particularly alginate or modified alginate chains. The polysaccharide,particularly alginate or modified alginate, chains may be included asside chains or auxiliary chains from a backbone polymer chain, which mayalso be a polysaccharide. Further, the polysaccharide chains may becrosslinked between side chains, auxiliary chains and/or backbonechains. These materials are advantageously modified by covalent bondingthereto of a biologically active molecule for cell adhesion or othercellular interaction. The materials are particularly useful to providepolymeric matrices for many applications, such as in tissue engineeringapplications for bone or soft tissue replacement. For example, the lossof bony tissue is a central feature of many aspects of clinicaldentistry (e.g. periodontal disease, caries, osteotomy for repair oftrauma) and matrices from the materials described herein can be usefulfor repair or replenishment of lost bony tissue. The materials are alsouseful for drug delivery applications when the biologically activemolecule is attached by a degradeable bond.

Unmodified alginate, a polysaccharide, has been previously utilized as atissue engineering matrix in cell encapsulation and transplantationstudies. It provides a useful matrix because cells can be immobilizedwithin alginate with little cell trauma and alginate/cell mixtures canbe transplanted in a minimally invasive manner. However, cells exhibitlittle or no adhesion or interaction with unmodified alginate. Oneaspect of this invention is to provide a matrix which combines specificcell adhesion ligands in the matrix such that high control overcell-matrix interactions, due to cell adhesion and matrix interactions,is attained.

One embodiment of the invention is directed to polymers containing apolymer backbone to which is linked polysaccharide groups, particularlyof alginates or modified alginates, which preferably are polymerizedD-mannuronate and/or L-guluronate monomers. The polysaccharide,particularly alginate, groups are present as side chains on the polymerbackbone which is intended to include side chains at the terminal end ofthe backbone, thus being a continuation of the main chain. The polymersprovide synthetic modified polysaccharides and alginates exhibitingcontrollable properties depending upon the ultimate use thereof.Further, the invention is directed to processes for preparing suchpolymers and to the use of such polymers, for example, as celltransplantation matrices, preformed hydrogels for cell transplantation,non-degradable matrices for immunoisolated cell transplantation,vehicles for drug delivery, wound dressings and replacements forindustrially applied alginates.

Another embodiment of the invention is directed to polysaccharides,particularly alginates, which are modified by being crosslinked. Thealginates may further be modified by covalent bonding thereto of abiologically active molecule for cell adhesion or other cellularinteraction. Crosslinking of the alginate can particularly providealoinate materials with controlled mechanical properties and shapememory properties which greatly expand their range of use, for example,to tissue engineering applications where size and shape of the matrix isof importance. The modification of the crosslinked alginates with thebiologically active molecules can provide a further three-dimensionalenvironment which is particularly advantageous for cell adhesion, thusmaking such alginates further useful as cell transplantation matrices.Further, the invention is directed to processes for preparing suchcrosslinked alginates and to their use, for example, for formingmaterials for tissue engineering and/or having cell adhesion propertiesparticularly for cell transplantation matrices, such as injectable celltransplantation solutions and preformed materials for celltransplantation.

Another embodiment of the invention is directed to modified alginates,such as alginate backbone (i.e. unmodified alginate) or the abovedescribed side chain alginates or crosslinked alginates, modified bycovalent bonding thereto of a biologically active molecule for celladhesion or other cellular interaction, which is particularlyadvantageous for maintenance, viability and directed expression ofdesirable patterns of gene expression. The modified alginate polymersprovide a three-dimensional environment which is particularlyadvantageous for cell adhesion. Further, the invention is directed toprocesses for preparing such polymers and to the use of such polymers,for example, for forming gels or highly viscous liquids having celladhesion properties particularly for cell transplantation matrices, suchas injectable cell transplantation solutions and preformed hydrogels forcell transplantation.

Further aspects of the invention may be determined by one of ordinaryskill in the art from the following description.

BACKGROUND OF THE INVENTION

Organ or tissue failure remains a frequent, costly, and serious problemin health care despite advances in medical technology. Availabletreatments now include transplantation of organs from one individual toanother, performing surgical reconstructing, use of mechanical devices(e.g., kidney dialyzer) and drug therapy. However, these treatments arenot perfect solutions. Transplantation of organs is limited by the lackof organ donors, possible rejection and other complications. Mechanicaldevices cannot perform all functions of an organ, e.g., kidney dialysiscan only help remove some metabolic wastes from the body. Likewise, druglevels comparable to the control systems of the body is difficult toachieve. This is partially due to difficulties in controlling the druglevel in vivo. Financially, the cost of surgical procedures is veryhigh. Advances in medical, biological and physical sciences have enabledthe emergency of the field of tissue engineering. “Tissue engineering”is the application of the principles and methods of engineering and thelife sciences toward the fundamental understanding of structure/functionrelationships in normal and pathological mammalian tissues and thedevelopment of biological substitutes to restore, maintain or improvefunction. It thus involves the development of methods to buildbiological substitutes as supplements or alternatives to whole organ ortissue transplantation . The use of living cells and/or extracellularmatrix (ECM) components in the development of implantable parts ordevices is an attractive approach to restore or to replace function. Theadvantage of this approach over whole organ/tissue transplantation isthat only the cells of interest are implanted, and they potentially canbe multiplied in vitro. Thus, a small biopsy can be grown into a largetissue mass and, potentially, could be used to treat many patients. Theincreased tissue supply may reduce the cost of the therapy because earlyintervention is possible during the disease, and this may prevent thelong-term hospitalization which results as tissue failure progresses.The use of immunosuppression may also be avoided in some applications byusing the patient's own cells.

Alginate is a linear polysaccharide, isolated, for example, from brownsea algae, which forms a stable hydrogel in the presence of divalentcations (e.g., Ba⁺⁺, Ca⁺⁺) (Smidsrod et al (1990): Alginate asimmobilization matrix for cells. TIBTECH, 8:71-78.) Alginate iscurrently being used for the in vitro culture of some cells types, as aninjectable cell delivery matrix, for immunoisolation based therapies,and as an enzyme immobilization substrate (Atala et al., 1993:Injectable alginate seeded with chondrocytes as a potential treatmentfor vesicoureteral reflux. J. Urology, 150:745:747; Levesque et al.,1992: Maintenance of long-term secretory function by microencapsulatedislets of Langerhans. Endocrinology, 130:644-650; Dominguez et al.,1988: Carbodiimide coupling of μ-galactosidase from Aspergillus oryzaeto alginate. Enzyme Microb. Technol., 10:606-610; and Lee et al. 1993:Covalent Immobilization of Aminoacylase to Alginate for L-hphenylalanineproduction. J. Chem. Tech. Biotechnol, 58:65-70.). Alginate hydrogelsare attractive for use with cells because of their mild gellingconditions, low diffusional barriers to cell nutrients, and lowinflammatory and nontoxicity in vivo (Smidsrod, supra).

Alginates occur naturally as copolymers of D-mannuronate (M) andL-guluronate (G) and have different monomer compositions when isolatedfrom different natural sources. The block length of monomer units,overall composition and molecular weight of the alginate influence itsproperties. For example, calcium alginates rich in G are stiffmaterials, (see Sutherland, I W (1991): Alginates. In Biomaterials.:Novel materials from biological sources.). It is theorized that gelformation is due primarily to the G-block, and that the M-block isessentially non-selective. In such arrangement, the calcium ions wouldbe selectively bound between sequences of polyguluronate residues andheld between diaxially linked L-guluronate residues which are in the ¹C₄chair conformation. The calcium ions would thus be packed into theinterstices between polyguluronate chains associated pairwise and thisstructure is named the “egg-box” sequence. The ability to form ajunction zone depends on the length of the G-blocks in differentalginates (Sutherland, supra.). Other advantages of alginates includetheir wide availability, low diffusional barrier for all nutrients andrelative biocompatibility (Smidsrod et al., Trends in Biotech, 8:71-78,1990).

A limitation of alginate hydrogels used with a cellular component is thelack of inherent cell adhesion. Such is necessary for cell attachmentand long term survival of most mammalian cell systems. Whilechrondrocytes and islets of Langerhans have been successfullytransplanted using alginates, the absence of suitable cell adhesion byalginates practically limits their use to cartilage and islet cellapplications. Most other cell types require attachment to anextracellular substrate to remain viable.

Previous attempts have been made to create a three-dimensional hydrogelenvironment incorporating cell adhesion ligands for cell attachment andsurvival. One system is a photopolymerizable polyacrylamide basedhydrogel with an RGD peptide grafted onto the polymer backbone. Thispolymer undergoes photogelation in the presence of UV light, and may bepolymerized as a polymer/cell hybrid (Moghaddam et al., 1993: Moleculardesign of three-dimensional artificial extracellular matrix:photosensitive polymers containing cell adhesive peptide. J. PolymerScience: Part A: Polymer Chem. 31:1589-1597.) Another is apolyacrylamide system, again with the RGD ligand covalently attached,which is catalytically polymerized prior to any biological interactions(Woerly et al. 1995: Intracerebral implantation of hydrogel-coupledadhesion peptides: tissue reaction. J. Neural Transplant. Plasticity,5:245:255.). A disadvantage of such systems is that conversion of thepolymers from a liquid to a solid, gel or highly viscous system requiresconditions which are detrimental to cell viability, e.g., use of organicsolvents and/or elevated temperatures.

Another major limitation of alginate hydrogels used in biotechnologyapplications is that their stability is dependent solely on calcium (orother divalent cation) binding, and this can present a limitation in theuse of these materials (e.g., loss of calcium from gels leads to geldissolution). In addition, alginate hydrogels have a limited range ofphysical properties due to the limited number of variables one cancurrently manipulate (i.e., alginate concentration, specific divalentcation used for gelling, and concentration of divalent cation). Thislimitation is especially evident when alginate is utilized as aninjectable cell delivery vehicle in tissue engineering. It is notpossible to obtain a pre-defined and desirable shape of the matrixfollowing injection, and it is thus not possible to create a new tissuewith a specific and desirable shape and size. This is especiallyimportant whenever the size and shape of the new tissue are critical tothe function of the tissue, for example, in reconstruction of facialfeatures such as nose or ears, or relining of joints.

SUMMARY OF THE INVENTION

An object of the present invention was to design improved syntheticanalogues of alginates, to provide a process for preparing such polymersand to provide compositions and methods utilizing such polymers,particularly in tissue engineering applications. It is further usefulaccording to the invention to provide alginate-containing materials inwhich the gel stability is related to an additional variable besidescation binding from the divalent cations. Thus, for example, thedisadvantages of the previous systems can be avoided by providing analginate which can be gelled or made highly viscous under mildconditions, i.e., in the presence of divalent metal cations such asCa⁺⁺or Ba⁺⁺in aqueous systems, without requiring, for example, organicsolvents and/or increased temperature.

In one embodiment, the invention provides polymers with side chains ofpolysaccharides in general which may not exhibit the gelling behavior ofalginates, but which provide polysaccharides with controllableproperties, such as degradation. These polymers may comprise a polymericbackbone section to which is covalently linked a polysaccharide sidechain. Another embodiment provides a polymeric backbone section to whichis bonded a side chain, preferably multiple side chains, of polymerized,optionally modified, D-mannuronate (M units) and/or L-guluronate (Gunits) monomers. The modified alginates preferably maintain the mildgelling behavior of conventional alginates, but do not have thedisadvantages discussed above. The linkage between the polymericbackbone section and the side chain(s) may be provided by difunctionalor multifunctional linker compounds, by groups incorporated within thepolymeric backbone section reactive with the polysaccharide units and/orby groups on the polysaccharide units or derivatives thereof reactivewith groups on the polymeric backbone section. The polymers mayadvantageously further comprise biologically active molecules bondedthereto, particularly preferably bonded through the carboxylic acidgroups on M and/or G units. In a particularly preferred embodiment, theside chains are alginates, the biologically active molecules exhibitcell adhesion properties and the polymers are useful for celltransplantation.

An advantageous aspect of these materials is the ability to provide apolymer analogous to alginates, but with high controllability of theproperties, particularly when used for cell transplantation purposes.The chemical structures, functionality and sizes of the different partsof the polymer, i.e., the backbone, linker, side chain and, optionally,biologically active molecule(s) can be provided so as to control manyproperties of the polymer in physiological systems, such as, forexample, degradeability, biocompatibility, organ or tissue specificityand affinity, cell adhesion, cell growth and cell differentiation,manner and rate of removal from the system, solubility and viscosity.

As the polymeric backbone section there can be used any homo- orco-polymer which is compatible with the ultimate use and which has theappropriate functional groups such that it can be covalently linkeddirectly or through a linker to the polysaccharide, particularlypolymerized M and/or G units, or suitable modifications thereof. Anypolymer meeting the above requirements is useful herein, and theselection of the specific polymer and acquisitions or preparation ofsuch polymer would be conventionally practiced in the art. See TheBiomedical Engineering Handbook, ed. Bronzino, Section 4, ed. Park.Preferred for such polymeric backbone section are, for example,poly(vinyl alcohols), poly(alkylene oxides) particularly poly(ethyleneoxides), polypeptides, poly(amino acids), such as poly(lysine),poly(allylamines) (PAM), poly(acrylates), modified styrene polymers suchas poly(4-aminomethylstyrene), polyesters, polyphosphazenes, pluronicpolyols, polyoxamers, poly(uronic acids) and copolymers, including graftpolymers thereof.

The polymeric backbone section may be selected to have a wide range ofmolecular weights, generally from as low as 100 up to ten million.However, by selection of the molecular weight and structure of thepolymeric backbone section the occurrence and rate of degradeability ofthe polymer and the manner and rate of release from physiologicalsystems of the polymer can be influenced. For instance, a high molecularweight non-degradable polymeric backbone section, for instance having amolecular weight above about 100,000, will in general provide a morestable polymer which may be useful in, for example, nondegradablematrices for immunoisolated cell transplantation. Alternatively, apolymeric backbone section having a molecular weight of less than about30,000 to 50,000 or one in which the backbone itself is degradable canbe cleared through the kidneys and by other normal metabolic routes.Polymers with a degradable polymeric backbone section include those witha backbone having hydrolyzable groups therein, such as polymerscontaining ester groups in the backbone, for example, aliphaticpolyesters of the poly(a-hydroxy acids) including poly(glycolic acid)and poly(lactic acid). When the backbone is itself degradable, it neednot be of low molecular weight to provide such degradeability. Aparticular example of a degradable polymer for the backbone is a graftpolymer of PEO (polyethylene oxide) and acetyl-aspartate shown by thefollowing equation, wherein the first equation shows formation of thedegradable polymer backbone, and the second schematic shows theattachment of side chains thereto:

A: DCC, HOBT, DMAP, DMF, B; X=OBT or OSu, NMM, DMF.

Copolymer content can be controlled by the block length of PEO andmixing in Ac-aspartate.

The solubility, viscosity, biocompatibility, etc., of the polymericbackbone section also is a consideration as to its effect on the desiredproperties of the final polymer product.

In one embodiment the polymeric backbone section can be one whichincorporates linkage sites for the polysaccharide side chains so that aseparate linker group is not required. For example, poly(amino acids)having free amino groups may be used for this purpose.

When a linker group is used, such linker group may be selected from anydivalent moieties which are compatible with the ultimate use of thepolymer and which provide for covalent bonding between the polymericbackbone section and the polysaccharide side chain(s). Such linkergroups are conventionally known in the art for such purpose, and can belinked to the backbone in a conventional manner. For linking of thelinker or linkage site on the polymer to the polysaccharide, since thepolysaccharide is generally bonded through a carboxylate group,chemistries useful for reacting with carboxylate groups are particularlyuseful in providing the linker or linkage site on the polymer; seeBronzino and Hermanson, cited below. The linker group may be selected tosignificantly affect the biodegradability of the polymer depending uponthe extent of hydrolyzability of groups in the linker chain. Amino acidlinkers and derivatives thereof are preferred due to the controllabilityof the degradation feature. For example, amino acid linker groups, suchas glycine, will provide ester linkages which are readily hydrolyzableand, thus, facilitate degradation of the polymer in an aqueousenvironment, whereas, amino alcohols provide an ether linkage which issignificantly less degradable. Amino aldehydes are also useful linkergroups. The substituent groups on the amino acids will also affect therate of degradeability of the linkage. The linker group may also bevaried in chain length depending upon the desired properties. Linkagesproviding, for example, from 1 to 10 atoms between the backbone and sidechain, are preferred, although longer linkage chains are possible.Further, the linker may be branched to provide multiple attachment sitesfor the side chains, for example, to provide a dendrite configurationsuch as shown in Example 5. The linker will be in the form of a residueof the linking compound without the group removed during bonding.

The side chains are polysaccharides, preferably optionally modifiedalginate units, which enable the preparation of a gel or highly viscousliquid in the presence of a divalent metal, e.g., Ca⁺⁺or Ba⁺⁺.Preferably they are comprised of polymerized D-mannuronate (M) and/orL-guluronate (G) monomers, but, also encompass modified such monomers.The side chains are particularly preferably comprised of oligomericblocks of M units, G units or M and G units. The molecular weight ofeach side chain or the number of units and length of such side chains isagain a function of the desired ultimate properties of the polymer andselectability of this aspect is an advantageous feature of theinvention. Although there is no specific limitation, the molecularweight of the side chain may range from about 200 up to one million, andmay contain, preferably 2 to 5,000 M and/or G units. As with thepolymeric backbone section, higher molecular weight side chains, e.g.above about 100,000, are generally useful when more stable polymers aredesired and lower molecular weight side chains, e.g., below about 30,000to 50,000, are generally useful when biodegradable species capable ofremoval through the kidneys, or other normal functions, are desired.

The distribution of M and G units also provides a controllabilityfeature of the invention with a higher ratio of G units generallyproviding a stiffer polymer which will hold its shape better. Sidechains having a percentage of G units based on the total of M & G unitsof from 10 to 100% are particularly preferred. Increasing or decreasingthe number of G units in the side chains will also allow for increasingor decreasing the rate of gelation of the polymer. Such may be ofinterest when the polymers are used in injectable solutions and the rateis controlled so that the solution will gel at the appropriate timeafter injection. The number of side chains provided on the polymericbackbone section also will affect the extent and rate of gelation and,thus, will vary depending on the ultimate use. In general, more sidechains will result in a more rigid, compact polymer, and provide a moredense concentration of attached biologically active molecules, ifpresent. The number of side chains is preferably from 1 to 100% of thereactive monomer units available on the backbone per polymer molecule.It is not necessary that every linker group or linkage site be providedwith a side chain. For example, free linkers or linkage sites may beleft to facilitate the solubility and/or compatibility of the polymer inits intended system. Additionally, free linkers or linkage sites may beprovided to allow for the later addition of differently structured orproportioned alginate side chains or other side chains.

Furthermore, the whole side chain or individual M and/or G units may bemodified from the naturally occurring units. Naturally occurring M and Galginate units exhibit the same general chemical structure irrespectiveof their source, although, the distribution and proportions of M and Gunits will differ depending upon the source. Natural source alginates,for example from seaweed or bacteria, can thus be selected to provideside chains with appropriate M and G units for the ultimate use of thepolymer. Isolation of alginate chains from natural sources for use asthe side chains herein can be conducted by conventional methods. SeeBiomaterials: Novel Materials from Biological Sources, ed. Byrum,Alginates chapter (ed. Sutherland), p. 309-331 (1991). Alternatively,synthetically prepared alginates having a selected M and G unitproportion and distribution prepared by synthetic routes analogous tothose known in the art can be used as the side chains. Further, eithernatural or synthetic source alginates may be modified to provide M and Gunits with a modified structure as long as the polymers with modifiedside chains still provide a gel or highly viscous liquid by interactionof the alginate units with a divalent metal. The M and/or G units may bemodified, for example, with polyalkylene oxide units of varied molecularweight such as shown for modification of polysaccharides in Spaltro(U.S. Pat. No. 5,490,978) with other alcohols such as glycols.Modification of the side chains with such groups generally will make thepolymer more soluble, which generally will result in a less viscous gel.Such modifying groups can also enhance the stability of the polymer.Further, the polymers can be modified on the side chains to providealkali resistance, for example, as shown by U.S. Pat. No. 2,536,893.

Useful polysaccharides other than alginates include agarose andmicrobial polysaccharides such as those listed in Table 1:

TABLE 1 Polymers^(a) Structure Fungal Pullulan (N) 1,4-;1,6-α-D-GlucanScleroglucan (N) 1,3;1,6-α-D-Glucan Chitin (N) 1,4-β-D-AcetylGlucosamine Chitosan (C) 1,4-β-D-N-Glucosamine Elsinan (N)1,4-;1,3-α-D-Glucan Bacterial Xanthan gum (A) 1,4-β-D-Glucan withD-mannose; D-glucuronic acid as side groups Curdlan (N) 1,3-β-D-Glucan(with branching) Dextran (N) 1,6-α-D-Glucan with some 1,2;1,3-;1,4-α-linkages Gellan (A) 1,4-β-D-Glucan with rhamose, D-glucuronic acid Levan(N) 2,6-β-D-Fructan with some β-2,1-branching Emulsan (A)Lipoheteropolysaccharide Cellulose (N) 1,4-β-D-Glucan ^(a)N-neutral, A =anionic and C-cationic.

The polymeric backbone section, linkages and side chains may be providedin a number of configurations which configuration will be a factor inthe controllability of the polymer properties. The configuration of thepolymeric backbone section, the number and location of linkage sites andthe type and number of side chains will determine the configuration.Examples of useful configurations are shown in FIG. 1 although theinvention is not limited to such configurations and furtherconfigurations using the three basic structural units can be providedaccording to the invention. Especially preferred, however, are polymershaving the branched configuration. It is noted that the “side chains” ofthe linear polymers are on the terminal ends of the backbone, but arestill considered side chains herein. Further, the side chains may bepresent between sections of polymer backbone in an alternating blocktype configuration.

One preferred embodiment is materials wherein the backbone itself is analginate. The side chains, for example, may be polyguluronate derivedfrom sodium alginate. A particular example involves cross-linkingpolyguluronate to itself, via a hydrolytically degradable bond,utilizing a bifunctional cross-linking molecule to form a cross-linkedpolymer. Dendritic polymers and comb polymers, as described below canalso be provided as such materials. These structures can provide ahighly cross-linked polymer which would rapidly degrade to low molecularweight components and readily be cleared by the body. To achieve thisgoal, for example, polyaldehyde guluronate is reacted with hydrazine andsodium borohydride to afford polyhydrazino guluronate. The hydrazinegroups on this alginate derived polymer are used to incorporate G-blockchains via the their hemiacetal termini. This provides materials fromnaturally derived polysaccharides with hydrolyzable hydrazone linkages,hence, biocompatible and biodegradable. Hydrolysis of the hydrazonelinkage in these materials will lead to short chain polysaccharides thatcan be excreted by the kidney. Further more, reduction of the hydrazonebond by borohydrides can form a chemically stable hydrazine bond thatprovide non-degradable materials. Thus, both biodegradable andnon-degradable biomaterials can be derived from natural polysaccharides.Cells within the polymer are not damaged by the cross-linking reaction,indicating that these materials are useful for cell transplantation, forexample.

Dendrimers provide a particularly interesting backbone structure sincethey exhibit different properties from the corresponding linear polymersdue to the difference in molecular shape and structures. Dendriticmolecules can be provided as a backbone with handles to branch off alarge number of functional groups in a compact region. Sincepolypeptides are biodegradable and their degradation products (i.e., theamino acids) are non-toxic, certain polypeptides (e.g. polylysines) canbe used as dendritic handles. In connection therewith, soluble polymersupports which combine the advantages of both solid phase and solutionphase syntheses can be used to prepare the materials. The most typicalsoluble polymer supports utilized are comprised of poly(ethylene oxide)(PEO). The reasons are the hydrophilic nature of PEO and insolubility ina variety of organic solvents which is desirable for purificationpurposes.

A further useful backbone structure is comb polymers which contain manyside chains extending from a polymer backbone. Poly(vinyl alcohol) (PVA)provides a particularly useful backbone for comb polymers. The alcoholgroups of the PVA can be esterified and subjected to the above-discussedcarbodiimide linkage chemistry to provide the side chain linkages.

The materials containing a polymer backbone may be prepared utilizingsynthetic methods known in the art, some of which are discussed above,for example in the Biomedical Engineering Handbook, section 4,; see alsoOdian, Principles of Polymerization, Chapter 9, 2nd ed., (1970). Forexample, polymeric backbone starting materials can be used which alreadycontain suitable linkage sites, e.g. free amino groups such as certainpoly(amino acids), or the polymers can be reacted with linker compoundsto provide suitable linkage sites, particularly by the reaction ofsuitable sites on the polymeric backbone with amino acid derivatives,optionally with the amino groups being protected. Further, some reactivesites on the backbone may be protected to prevent addition of the linkergroup if it is desired to keep such sites free or to subsequentlyprovide such sites with different linker groups. This chemistry isconventional in the field of linker/polymer formation, especiallyinvolving ester, amide, ether and other covalent linkages; see, e.g.,Bronzino and Hermanson, cited above. For protective groups, see, e.g.,Vogel's Textbook of Practical Organic Chemistry, 5th ed. p. 550+ and784+. After removal of the optional protecting groups on the linker,reaction with the side chain of M and/or G units is conducted,preferably through grafting by reductive amination of the reducing endof the side chain with the amino group of the linker, to produce thesubject polymers. The side chains are provided as described above fromnatural sources or synthetically, and may have, optionally, thedescribed modifications they may be bonded as described above, or byother conventional methods.

Another embodiment of synthetic analogues of alginate materials arethose provided by covalent crosslinking of the alginate. This covalentcrosslinking greatly expands the range of situations in which thesematerials are useful. One specific application of this modification isthe development of matrices of the alginate with shape memory. Thecrosslinked alginate provides advantageous shape memory properties andcompression resistance properties which make them particularlyadvantageous for use in forming cell transplantation matrices. Shapememory matrices are designed to “remember” their original dimensionsand, following injection in the body in a compact form (e.g., through asyringe) or other means of placement in the body or in other locationswhich they may find use, resume their original size and shape. The shapememory property of the alginate is provided by crosslinking thereof.Crosslinking can also improve the compression resistance and/or othermechanical properties of the alginate. Further, a crosslinked alginatecan provide a degree of cell adhesion even without use of biologicallyactive cell adhesion ligands. Gelling by divalent cations providesanother means of increasing the viscosity and degree of structure of thealginate in addition to the crosslinking. Further, the crosslinkedalginate may be covalently bonded to at least one cell adhesion ligandto provide for cell adhesion and maintenance of cell viability.

It is also an object of the invention to provide a process for preparingsuch crosslinked alginates and to provide compositions and methodsutilizing such crosslinked alginates.

The alginate used for crosslinking according to the invention arealginate chains which contain polymerized D-mannuronate (M) and/orL-guluronate (G) monomers, but the term “alginate” or “alginate chain”as used herein also is intended to encompass chains wherein suchmonomers are modified such as described below when they are compatiblewith the ultimate use and able to be crosslinked covalently. Thealginate chain is particularly preferably comprised of oligomeric blocksof M units, G units, M and G units, or mixtures of such blocks. Thegeneral structure of an alginate linear copolymer of M and G units isdemonstrated by the following general formula:

The molecular weight of the alginate chain and, thus, the number ofunits and length of the chains may be selected dependent upon thedesired properties of the polymer. In general, the molecular weight ofeach chain may range from about 1,000 to one million, for example.Higher molecular weight chains, e.g., above about 100,000, are generallyuseful when more stable alginate polymers are desired and lowermolecular weight chains, e.g., below about 30,000 to 50,000, aregenerally useful when biodegradable species capable of removal throughthe kidneys or through other normal metabolic functions are desired.

The distribution of M and G units also provides a controllabilityfeature of the invention with a higher ratio of G units generallyproviding a stiffer alginate material which will hold its shape better.An alginate chain having a percentage of G units based on the total of Mand G units of from 10 to 100% is particularly preferred. Increasing ordecreasing the number of G units in the chain will also allow forincreasing or decreasing, respectively, the rate of gelation of thealginate. Such may be of interest when the alginate is used in aninjectable solution and the rate is controlled so that the solution willgel at the appropriate time after injection.

The alginate chain or individual M and/or G units may also be modifiedfrom the naturally occurring units. Sources for the naturally occurringalginates and for modified alginates are described above in relation tothe alginate side chains for the polymeric backbone embodiment describedabove.

Furthermore, useful as the alginate starting material are materialshaving a polymeric backbone to which is linked alginate side chains, asdescribed above. The crosslinking may occur between side chains of thesame backbone and/or between side chains of other backbones. It is alsopossible to have different types of alginate-containing materials withcrosslinking provided between alginate sections or chains thereof.Mixtures of any of the above alginate starting materials may also beused.

The crosslinking of the alginates is by action of a crosslinking agentto provide covalent bonding, through the crosslinking agent, from thecarboxylic acid groups of the uronic acid of one alginate unit to thecarboxylic acid group of the uronic acid of another alginate unit. Suchcrosslinking is preferably between alginate units from differentalginate chains. However, crosslinking may also occur between alginateunits of the same chain or, in the case where the alginates are sidechains on a polymer backbone as described above, crosslinking may occurbetween different side chains on the same or differing polymerbackbones.

The crosslinking agent may be any suitable agent with at least twofunctional groups which are capable of covalently bonding to thecarboxylic acid groups and/or alcohol groups of the alginate or modifiedgroups therefrom. Crosslinking agents of higher functionality may alsobe used. For example, polyamines such as bifunctional, trifunctional,star polymers or dendritic amines are useful and these can be made, forexample, by conversion from corresponding polyols. Preferredcrosslinking agents are those with at least two nitrogen-basedfunctional groups such as, for example, diamine or dihydrazidecompounds; non-limiting examples thereof being diamino alkanes,Jeffamine series compounds, adipic acid dihydrazide and putrescine.Particularly preferred as a crosslinking agent is lysine, especially anester thereof, particularly the methyl or ethyl ester.

The crosslinking agent may also be selected to provide a more or lessbiodegradable or non-biodegradable bond such that the lifetime of theresulting crosslinked alginate material in its environment, e.g. invivo, can be modified for the intended utility.

The amide bonds formed when crosslinking with an amine crosslinkingagent of alginates are less susceptible to hydrolytic cleavage comparedto the acetal linkages between the consecutive uronic acids units ofalginates. Therefore, products crosslinked with regular diamines are ofrelatively low biodegradability in this series of materials, since thepolysaccharide (alginate) will degrade before the linking moleculeswill. To improve upon the rate of biodegradation, a more labilefunctional group may be incorporated into the crosslinker. Bifunctionalbiodegradable crosslinkers may be synthesized according to wellestablished chemical pathways. See the following schematic exemplifyingpreparation of a crosslinking agent with biodegradable ester linking:

Modification of ethylene glycol to form biodegradable bifunctionalcrosslinkers. For example, ethylene glycol could be coupled with twoN-(t-Boc)glycine using carbodiimide chemistry to yield1,2-ethylene-(N,N′-di-t-Boc)glycine intermediate. This intermediatecould be deprotected using trifluoroacetic acid in methylene chloride atvarious temperatures to yield 1,2-ethyleneglycoldiglycinateintermediate. This intermediate could be deprotected usingtrifluoroacetic acid in methylene chloride at various temperatures toyield 1,2-ethyleneglycoldiglycinate intermediate. In addition toethylene glycol, other molecules with two terminal alcohol functionalgroups could be utilized. Moreover, polyols including, e.g., (starshaped or dendritic) could be transformed into similar types ofcrosslinkers with biodegradable ester functional groups incorporatedusing parallel chemical pathways.

Preferably, though not necessarily, the crosslinking is facilitated byan activator compound which reacts with the carboxylic acid group of thealginate unit to make it more reactive to the crosslinking agent. Usefulactivators for making a carboxylic acid group more reactive to thecrosslinking agent, particularly an amine functional group of thecrosslinking agent, are known in the art. Examples thereof include, butare not limited to, carbodiimides, particularly water-solublecarbodiimides such as, for example,1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC),1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide (CMC), and CDI(carbodiimidazole).

Also preferred, when using an activator compound, is the use of astabilizer for stabilizing the resulting activated group. Again, usefulstabilizers for the activator groups are known in the art. Forcarbodiimides, particularly EDC, a useful stabilizer is1-hydroxybenzotriazole (HOBT) which stabilizes the activated groupagainst hydrolysis. Other useful stabilizers includeN-hydroxysuccinimide and N-hydroxysulfylsuccinimide (sulfo-NHS).

The reaction sequence using a lysine ethyl ester crosslinking agent withEDC activator and HOBT stabilizer is shown in the following schematic:

Reaction pathway of alginate crosslinking. EDC activate carboxylic acidto yield O-acylisourea intermediate. This intermediate reacts with HOBTto form HOBT activated intermediate. Primary amino groups in lysineethyl ester then couple the activated carboxyl groups of adjacentalginate molecules to form crosslinked alginates.

The crosslinking can generally be conducted at room temperature andneutral pH conditions, however, the conditions may be varied to optimizethe particular application and crosslinking chemistry utilized. Forcrosslinking using the EDC chemistry, optionally with HOBT or sulfo-NHSintermediate steps, pH of from 4.0 to 8.0 and temperatures from 0° C. toroom temperature (25° C.) are optimal and preferred. It is known thathigher temperatures are unpreferred for this chemistry due todecomposition of EDC. Similarly, basic pH (e.g., 8-14) is alsounpreferred for this reason when using this chemistry.

Other crosslinking chemistries can also be used. For example, usingpoly(ethylene glycol) (PEG) as a spacer in a crosslinking agent with anN-protected amino acid (see Example 12). Also, crosslinking of oxidizedalginate can be conducted with adipic acid dihydrazide. The oxidationresults in polyaldehyde alginates (limit oxidized alginates) forcrosslinking (See Example 17). Additionally, crosslinking can beeffected by light activation using photoreactive materials (See Example26).

Another method of altering the mechanics of crosslinked systems is byvarying the molecular weight between cross-links, M_(c), in the polymernetwork (Peppas and Bar-Howell, Hydrogels in Medicine and Pharmacy. Vol1, CRC, Boca Raton, pp 28-55, 1986; and, Anseth et al., Biomaterials17:1647-1657, 1996). M_(c) may be modified by controlling the extent ofcross-linking, or by varying the molecular weight of the cross-linkingmolecule (Simon et al., Polymer 32:2577-2587, 1991). Both of thesestrategies may be utilized to alter the mechanical properties of thealginate gels. Covalent cross-linking has been achieved with severaldifferent approaches. Cross-linking with lysine results in amide bondformation, which will provide stability and will degrade very slowly.PEG-crosslinkers contain an ester bond, which will be more labile tohydrolysis. Finally, cross-linking of oxidized alginate with adipic aciddihydrazide leads to a hydrazone bond. Importantly, these materials maybe both covalently and ionically (e.g., calcium) cross-linked. This mayprove advantageous in certain applications in which one desires atwo-stage gelling. For example, the polyaldehyde alginates describedbelow will cross-link ionically very quickly (e.g., minutes), while thecovalent cross-linking reaction can be designed to occur very slowly(e.g., hours). A surgeon could thus ionically cross-link these polymersto yield a solution which is amenable to injection via a syringe orendoscope, but is viscous enough (viscosity at this stage decreases withincreasing extent of oxidation) so that it does not extravasate afterbeing placed. The covalent cross-linking would subsequently harden theimplanted material into a more rigid, non-flowable mass.

Crosslinking of the alginate provides a more structured material, theextent of structuring being dependent, at least in part, on the extentof crosslinking. The extent of structuring of the alginate material willalso depend, among other factors, upon the extent of gelling throughaction of the ionic bonding of the divalent metal cation, as discussedabove, and upon the nature of the starting alginate material, which asdiscussed above may be varied, for example, to affect stiffness of thematerial. Depending on the extent of crosslinking and these otherfactors, the crosslinked alginate material may run the spectrum throughthe following forms: a viscous liquid, a swellable gel, a non-swellablegel, a swollen polymer network or a solid matrix, for example.

The extent of crosslinking is a function of the amount of crosslinkingagent and crosslinking method used, i.e., the molar percent ofcrosslinking agent per mole of crosslinkable alginate carboxylic acidgroups. The alginate will be increased in viscosity as it iscrosslinked. Thus, the extent of crosslinking will be dependent upon theultimate use. For example, to provide gel materials which have superabsorbency properties, it is useful to have a low crosslinking extent,for example, of about 1 to 20%, preferably 1-10%, of crosslinkablegroups crosslinked. For tissue matrix materials, for example, the extentof crosslinking is preferably from about 5% to 75%. In a particularembodiment described in the following examples, the alginate is aviscous liquid when the crosslinking agent amount is about 25 mol % orless, a swellable gel when the amount is about 50% and a solid structurewhich maintains its size and shape when the amount is about 75% orhigher. However, the crosslinking chemistry can be selected andoptimized to control viscosity even at lower crosslinking extent. Inanother embodiment, the crosslinking agent is used in a molar amountabout equal (i.e., 100 mol %) to the number of crosslinkable alginatecarboxylic acid (uronic acid) groups.

Additionally, the crosslinking can be conducted either before, after orsimultaneously with the gelling by action of the divalent metal cations.It is preferred for certain applications that the crosslinking beconducted either before or simultaneously with the gelling by divalentcation so as to prevent problems with diffusion of the crosslinkingagent to interior portions of the gelled material.

This material may make an ideal two-stage gelling matrix. The extent ofoxidation of the alginate in the first step of the synthesis controlsthe binding sites available for ionic gelling, and thus regulates theviscosity of calcium cross-linked gels. The covalent cross-linkingreaction with adipic acid dihydrazide occurs over several hours, andthus can be used to harden the gel slowly. The ultimate mechanicalproperties of the matrix can be controlled by varying the extent ofcovalent cross-linking, and this will be a function of the adipic acidconcentration. For example, a material largely insensitive to the timeof ionic cross-linking time, and with a time frame for ioniccross-linking considerably shorter than that for covalent cross-linkingcan be designed.

In a further embodiment, the crosslinked alginate is not gelled byaction of divalent metal cations at all or is gelled by cations presentin vivo only after the delivery of the crosslinked alginate into thebiological system, e.g., body.

For the reasons discussed above, the extent of stiffness and matrixstructure of the crosslinked alginate materials will be influenced bothby the gelling by divalent cation and by the extent and nature ofcrosslinking. The ability to vary these and other factors provides greatflexibility in designing a material which is particularly suited for itsultimate application.

In addition to the type of cell adhesion discussed below, the matrixstructure provided by the crosslinked alginates themselves canfacilitate cell adhesion type properties, for example, due to trappingof cells in the matrix or action of a crosslinking agent, such aslysine. For example, the crosslinked alginate as a matrix can beintroduced for tissue engineering and the cell can migrate into thepores of the matrix in vivo. It is also advantageous, however, toprovide the crosslinked alginates with biologically active molecules tofacilitate cell adhesion or other biological interaction, as discussedbelow. The ligands may be added before, during or after crosslinking ofthe alginate and/or gelling by divalent cations.

To address the relative biological inertness of the syntheticallymodified polysaccharide or alginate materials discussed above, thepolymers can be modified with biologically active molecules. Anotheraspect of the invention lies in modifying not only the above-discussedsynthetic alginate analogues but also the base naturally occurring,modified or analogous alginate materials which are described herein.Even if the alginate or modified alginate material is not provided on apolymeric backbone and/or not crosslinked, the coupling of the alginatewith certain biologically active molecules makes it very useful fortissue engineering and other applications.

The polymeric backbone-containing and/or crosslinked alginates and thenaturally occurring or modified base alginate materials can be modifiedwith the cell adhesion active molecule(s), for example, by covalentbonding using amide chemistry between the amine groups of the biologicalmolecules and a free carboxylic acid group of the uronic acid residues(of M and G units) of the alginate or other polysaccharide. If thematerial is crosslinked, bonded to a polymeric backbone and/or otherwisemodified, free acid groups must remain to add cell adhesion groups. Ifthe cell adhesion groups are added first, active groups for anysubsequent crosslinking, polymer bonding or other modification mustremain. Other chemistries can also be used to effect such bonding to thebiologically active molecule. For example, alginate or analogousmaterials can be modified to provide aldehyde groups thereon, which arereactive with the amino terminal of peptides to provide an imine bondwhich is reduced to a stable amine bond. An example of this chemistry isdescribed in Example 24 herein.

Examples of suitable cell adhesion molecules include known cellattachment peptides, proteoglycan attachment peptide sequences (seeTable 2), biologically active proteoglycans (e.g. laminin andfibronectin) and other polysaccharides (e.g., hyaluronic acid andchondroitin-6-sulfate). Examples of other suitable biological moleculesinclude peptide growth factors (such as EGF, VEGF, b-FGF, acidic FGF,platelet-derived growth factor, TGF or TGF-β), and enzymes (Dominguez etal., 1988: Carbodiimide coupling of β-galactosidase from Aspergillusoryzae to alginate. Enzyme Microb. Technol., 10:606-610; and Lee et al,1993: Covalent Immobilization of Aminoacylase to Alginate forL-h\phenylalanine production. J. Chem. Tech. Biotechnol, 58:65-70).Examples of these molecules and their function are shown in thefollowing Table 1.

TABLE 1 Proteins specific for cell binding from extracellular matrix.From Hubbell, JA (1995): Biomaterials in tissue engineering.Bio/Technology 13:565-576. One-letter abbreviations of amino acids areused, X stands for any amino acid. Protein Sequence Role FibronectinRGDS Adhesion of most cells, via α,β₁ LDV Adhesion REDV AdhesionVitronectin RGDV Adhesion of most cells, via α,β₁ Laminin A LRGDNAdhesion IKVAV Neurite extension Laminin B1 YIGSR Adhesion of manycells, via 67 kD laminin receptor PDSGR Adhesion Laminin B2 RNIAEIIKDANeurite extension Collagen 1 RGDT Adhesion of most cells DGEA Adhesionof platelets, other cells Thrombospondin RGD Adhesion of most cells VTXGAdhesion of platelets

TABLE 2 Amino acid sequences specific for proteoglycan binding fromextracellular matrix proteins. From Hubbell, above. PROTEIN SEQUENCEXBBXBX* Consensus sequence PRRARV Fibronectin YEKPGSPPREVVPRPRPGVFibronectin RPSLAKKQRFRHRNRKGYRSQRGHSRGR Vitronectin RIQNLLKITNLRIKFVKLaminin

Particularly preferred as the cell adhesion molecule bonded to thealginate chain are synthetic peptides containing the amino acid sequencearginine-glycine-aspartic acid (RGD) which is known as a cell attachmentligand and found in various natural extracellular matrix molecules.Further of interest is GREDVY (endothelial cell specific) peptide. Thealginates with such a modification provide cell adhesion properties tothe alginate analogue, natural alginate or modified alginate,particularly when used as a cell transplantation matrix, and sustainslong-term survival of mammalian cell systems, as well as controllingcell growth and differentiation.

Coupling of the cell adhesion molecules to the alginate can be conductedutilizing synthetic methods which are in general known to one ofordinary skill in the art. A particularly useful method is by formationof an amide bond between the carboxylic acid groups on the alginatechain and amine groups on the cell adhesion molecule. Other usefulbonding chemistries include those discussed in Hermanson, BioconjugateTechniques, p. 152-185 (1996), particularly by use of carbodiimidecouplers, DCC and DIC (Woodward's Reagent K). Since many of the celladhesion molecules are peptides, they contain a terminal amine group forsuch bonding. The amide bond formation is preferably catalyzed by1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), which is a watersoluble enzyme commonly used in peptide synthesis. An example of suchchemistry is shown in the following equation.

Therein, EDC reacts with carboxylate moieties on the alginate backbonecreating activated esters which are reactive towards amines. R-NH₂represents any molecule with a free amine (i.e. lysine or any peptidesequence N-terminus). To reduce unfavorable side reactions, EDC may beused in conjunction with N-hydroxysuccinimide,N-hydroxysulfylsuccinimide or HOBT to facilitate amide bonding overcompeting reactions.

The reaction conditions for this coupling chemistry can be optimized,for example, by variation of the reaction buffer, pH, EDC:uronic acidratio, to achieve efficiencies of peptide incorporation between 65 and75%, for example. Preferably, the pH is about 6.5 to 7.5. The ionicconcentration providing the buffer (e.g. from NaCl) is preferably about0.1 to 0.6 molar. The EDC:uronic acid groups molar ratio is preferablyfrom 1:50 to 20:50. When HOBT is used, the preferred molar ratio ofEDC:HOBt:uronic acid is about 4:1:4. The density of cell adhesionligands, a critical regulator of cellular phenotype following adhesionto a biomaterial. (Massia and Hubbell, J. Cell Biol. 114:1089-1100,1991; Mooney et al., J. Cell Phys. 151:497-505, 1992; and Hansen et al.,Mol. Biol. Cell 5:967-975, 1994) can be readily varied over a 5-order ofmagnitude density range. An example thereof is shown in FIG. 2.

Both surface coupling, as well as bulk coupling of alginate can bereadily obtained with this coupling chemistry. Therefore, bymanipulation of surface and bulk coupling, materials having one type ofmolecule coupled internally in the matrix and another type of moleculecoupled on the surface can be provided, for example.

Other methods conventionally known for attachment or immobilization ofadhesion ligands may be used, such as discussed in Bronzino cited above,p. 1583-1596.

The biological molecules useful for attachment to the above-describedalginate materials are not, however, limited to those providing celladhesion function. For example, the polymer could be bound to a moleculewith antiseptic function when used as a wound dressing, or whichprovides adhesion tissue specific gene expression, growth factors toenhance proliferation of cells in the environment or vascularation ofthe tissue or anti-inflammatory activity.

The combination of the alginate and alginate analogue materials withcell adhesion ligands bonded thereto provides a unique three dimensionalenvironment in which the cells interact through various forces foradhesion to the alginate which has many uses, particularly for tissueengineering applications. The cell adhesion ligands provide specificcell membrane receptor sites for the desired cells. The number, type andlocation of the cell adhesion ligands on the alginate or alginateanalogue material will affect the cell adhesion and cell viabilitymaintenance properties and such factors can be varied to suit theparticular application. Such applications include tissue engineeringmethods applied to humans and animals. Preferably, 10⁻¹² to 10⁻⁴ molesof adhesion molecules per milliliter of hydrated alginate are used; seeMassia et al., J. Cell. Biol; Vol. 114, p. 1089-1100 (1991). Also,combinations of the cell adhesion ligands with differing cell adhesionligands or other bioactive molecules may be utilized according to theinvention. Such additional groups may be bonded at other sites on thealginate or to suitable sites on ligands already present on the alginateor alginate analogue material.

The alginate having a polymeric backbone and/or being crosslinked or thenatural or modified alginate or other polysaccharide, optionally withbioactive molecules, can create a synthetic extracellular envirornentfor mammalian cells that is capable of performing the diverse functionsof the natural extracellular matrix (ECM). The materials describedherein will, thus, have application in the field of tissue engineering,biomaterials, and in the basic cell biological sciences for studyingthree dimensional cell interactions and tissue morphogenesis. Thematerials described herein are advantageous as a model system forcreating a synthetic ECM capable of guiding cellular gene expressionduring in vitro or in vivo tissue formation.

The natural ECM regulates cell growth and differentiation with featuresthat allow the control of the mechanical and chemical environment aroundthe cells (D. E.

Ingber. Mechanochemical Switching between growth and Differentiation ba,Extracellular Matrix, in Principles of Tissue Engineering (Ed, Lanza,Langer and Chick) p. 89-100 (1997)). The alginate and analogue materialsare capable of displaying a wide range of mechanical properties and,with covalent modification by the bioactive molecules as described, candisplay a wide range of biochemical properties, such as connectingmammnalians cell with the extracellular environment which previous cellencapsulation matrices have not been capable of. The covalentmodification with bioactive sequences allows the creation of atwo-dimensional or three dimensional synthetic extracellular environmentcapable of providing biochemical signaling in the form of sequesteredgrowth factors, hormones or active sequences within these specificchemicals, and more importantly it will allow mammalian cells tocommunicate with other cells directly through the alginate material viacell attachment peptides (e.g., RGD, YIGSR, REDV) covalently attacheddirectly to the material. By then controlling the mechanical propertiesof the alginate material—for example by the nature of the polymerbackbone and/or by crosslinking and/or by modifications of the alginatechain thereof in the manners discussed above—it will be possible tocontrol the intercellular signaling between the cells and among cellpopulations (see D. E. Ingber, Mechanochemical Switching between Growthand Differentiation by Extracellular Matrix, in Principles of TissueEngineering (Ed. Lanza, Langer and Chick )p. 89-100 (1997) and G FOster, J D Murray, and A K Harris, Mechanical aspects of MesenchymalMorphogenesis, Journal of Embryology and Experimental Morphology, Vol.78, p. 83-125 (1983)).

Unmodified alginate has been used as a cell immobilization material formany years due to the stable hydrogels formed with mild gellingconditions. However, the alginate acts only as a neutral agentsuspending cells or cell aggregates in three dimensions. By modifyingthis polysaccharide structurally in the manners discussed above andoptionally with cell attachment peptides, growth factors, hormones orECM binding sequences, for example, the alginate can be transformed intoa dynamic, interactive matrix capable of guiding cellular geneexpression in space and time. The ability to control the viscoelasticproperties of the alginate is an integral aspect in guiding cellulargene expression (see M. Opas, Substratum Mechanics and CellDifferentiation. International Review of Cytology, Vol. 150, p. 119-137(1994); and G F Oster, J D Murry, and A K Harris, Mechanical aspects ofMesenchymal Morphogenesis, Journal of Embryology and ExperimentalMorphology, Vol. 78, p 83-125 (1983)) and can be used in model in vitrocell culture systems and tissue engineering applications.

Matrices play a central role in tissue engineering. Matrices areutilized to deliver cells to desired sites in the body, to define apotential space for the engineered tissue, and to guide the process oftissue development. Direct injection of cell suspension without matriceshave been utilized in some cases, but it is difficult to control theplacement of transplanted cells. In addition, the majority of mammaliancell types are anchorage dependent and will die if not provided with anadhesion substrate.

Alginate materials in polymerized form and/or crosslinked and/ormodified with bioactive molecules, as discussed above, can beadvantageously used as matrices to achieve cell delivery with highloading and efficiency to specific sites. The materials according to theinvention also provide mechanical support against compressive andtensile forces, thus maintaining the shape and integrity of the scaffoldin the aggressive environments of the body. This is particularly thecase when the alginate is crosslinked to a higher degree. The scaffoldprovided by these materials may act as a physical barrier to immunesystem components of the host, or act as a matrix to conduct tissueregeneration, depending on the design of the scaffold.

The first type of scaffolds, immunoprotective devices, utilize asemipermeable membrane to limit communication between cells in thedevice and the host. The small pores in these devices, e.g., (d<10 μm)allow low molecular weight proteins and molecules to be transportedbetween the implant and the host tissue, but they prevent large proteins(e.g., immunoglobulins) and host cells (e.g., lymphocytes) of the immunesystem from entering the device and mediating rejection of thetransplanted cells. In contrast, open structures with large pore sized,e.g., (d>10 μm) are typically utilized if the new tissue is expected tointegrate with the host tissue. The morphology of the matrix can guidethe structure of an engineered tissue, including the size, shape andvascularization of the tissue.

As discussed above, the alginate, alginate analogue and modifiedalginate materials of the invention are useful for cell transplantationmatrices. These materials can be used to provide such a matrix in any ofseveral ways. For instance, when the matrix is desired to be a temporarymatrix for replacement by natural tissue, the material can be designedfor biodegradability and system release, for example, by providinghydrolyzable linkages, using relatively low molecular weight alginatechains, biodegradable crosslinking agents, biodegradeable polymerbackbones and/or low molecular weight polymer backbone sections.Alternatively, when less degradable matrices are desired,non-hydrolyzable linkages, alginate chains of higher molecular weight,non-degradable crosslinking agents and/or higher molecular weightpolymer backbone sections can be used. The many ways in which theproperties of the materials can be altered provides a high degree ofcontrollability in providing materials which meet the requirements forthe specific application.

In a less degradable form, the matrices can be introduced to the bodywithout cells, but cells will migrate into the matrix, in vivo, andregenerate therein. The alginate or analogue material can be provided inan injectable form, optionally bound to appropriate viable cells, afterinjection in which case endogenous divalent metal cation in thephysiological system after injection causes gelation of the alginateportions of the material. Alternatively, divalent metal cations areadded to the solution, for example as a calcium sulfate solution, justprior to injection. As discussed above, the material can be designed tocontrol its rate of gelation to match the ultimate utility. Suchinjectable solutions can be utilized for delivery of cells to regenerateurologic tissues, for reconstructive surgery, skin replacement, otherorthopedic applications or other tissue replacement or repairapplications. The alginate-containing materials provide a highlystructured, gelled or highly viscous matrix in which the cells arecompatible and grow to achieve their intended function, such as tissuereplacement, eventually replacing the matrix.

As such, the materials, particularly the polymeric type, may act asanalogs to natural glycosamine-glycans and proteoglycans of theextracellular matrix in the body. Furthermore, they can be used toprovide preformed gelled or highly viscous matrices bound to cells whichmay then be surgically implanted into a body. It is of particularlysurprising advantage that the materials can be used to implant a matrixwhich does not contain cells and subsequently the cells can be seededinto the matrix in vivo. The materials optionally may be provided, forexample, as a gel, as a viscous solution, as a relatively rigid body, aspreformed hydrogel, within a semi-permeable membrane, withinmicrocapsules, etc., and the polymer properties controlled as discussedabove to facilitate such applications. The utility of the polymers forcell transplantation and tissue engineering is a significant advance inthe art, particularly since it was previously considered not to bepractical or possible to achieve such results with synthetic materials;see C. Ezzell, The Journal of NIH Research, July 1995, Vol. 7, p. 49-53.

The materials are also advantageously useful as vehicles for drugdelivery particularly for sustained release. For drug deliveryapplication, it is useful that the bioactive molecule, i.e., the drug,be linked to the alginate polymer and/or analogue material by adegradeable bond chosen for controllable release in the system. Otherutilities of the materials which may or may not employ a boundbiological molecule include, for example, wound dressings, wound healingmatrix materials, matrices for in vitro cell culture studies,replacements for conventional industrial alginates used, for instance,as food thickening agents and as printing additives, for example tothicken inks, and similar uses wherein the above-described propertiesare desired. One particularly advantageous use of the crosslinkedmaterials, not necessarily containing bioactive components, is as highlyabsorbent materials. Particularly, materials with a low extent ofcrosslinking, e.g., about 1-20% crosslinking, have this utility. Theabsorbency property makes them useful, for example, in disposable diaperapplications. The controllability of the properties of the syntheticpolysaccharides according to the invention and the consistentreproduceability of such selected synthetic polysaccharides makes themparticularly advantageous for many applications.

The entire disclosure of all applications, patents and publications,cited above and below, is hereby incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides examples of useful configurations of the polymericbackbone sections with side chains.

FIG. 2 provides an example that the density of cell adhesion ligands canbe readily varied over a 5-order of magnitude density range.

FIG. 3 provides an example of the adhesion of 3T3 fibroblasts andskeletal myoblasts to alginate matrices in culture.

FIG. 4 shows alginate gels cross-linked with lysine and cut into slabsfor testing their shape memory.

FIG. 5 shows scanning electron microscopy examination of crosslinkedalginate matrices revealing a highly porous material with large pores.

FIG. 6 is a graph showing that the mechanics of the hydrogel network canbe controlled by varying the amount of lysine available forcross-linking.

FIG. 7 is a graph showing that the swelling ability of lysinecrosslinked alginate hydrogels decreases with increased crosslinking.

FIG. 8 is a graph showing the elastic modulus in compression [KPa]versus number of repeat units in cross-linking molecule.

FIG. 9 is a graph showing elastic modulus vs. cross-link density [%]utilizing the PEG of 1000 molecular weight as the cross-linkingmolecule.

FIG. 10 is a graph showing the compressive modulus of limit-oxidizedalginates cross-linked with adipic dihydrazide at various % w/walginates.

FIG. 11 is a graph showing that the mechanical strength of cross-linkedlimit-oxidized alginates depends on the concentration of thecross-linker as well as the calcium ion content in the final gel.

FIG. 12 is a graph showing a significant increase in the compressivemodulus as the calcium ion concentration increased.

FIG. 13 is a graph for gels cross-linked at various concentrations ofpolyaldehyde guluronate wherein the compressive modulus was measured andplotted against final % wlw PAG.

FIG. 14 is a graph of the compressive modulus versus adipic dihydrazideconcentration.

FIG. 15 is a graph showing that the compressive modulus of the gelsincreased with increasing calcium concentrations.

FIG. 16 is a graph showing that by adjusting the pH a change in thecompressive modulus was observed.

FIG. 17 is a graph showing that the degree of cross-linking in thesematerials can also be controlled by varying the degree of oxidation ofthe polyguluronate chains.

FIG. 18 is a graph of the % of VEGF release over time.

FIG. 19 is a graph of the % of VEGF release over time for a differentsystem.

FIG. 20 shows the degree of coupling for certain species.

EXAMPLES

All temperatures are set forth uncorrected in degrees Celsius; and,unless otherwise indicated, all parts and percentages are by weight.

Example 1

The following scheme demonstrates one method of preparation of anembodiment of the inventive alginates modified with the cell adhesionmolecule RGD:

In this reaction scheme for alginate/peptide conjugation, carbodiimideenzyme (EDC) is added to 0.5% alginate solutions. Fifteen (15) minutesare allowed for activation of the carboxylic acid groups on the alginatebackbone. The RGD containing peptide is added to the reaction andallowed to react for 24 hours at room temperature. The reaction isquenched by the addition of 1N HCL which deactivates the EDC. Thesolution is brought back to pH 7 with addition of 1M NaOH andextensively dialyzed over 3 to 5 days, removing unreacted chemicals. Thepolymer is then redissolved in water and sterile filtered.

Example 2

A pentapeptide (GRGDY) was used as the model cell adhesion peptide. TheN-terminal free amine provides a site for coupling to alginate, whilethe C-terminal tyrosine provides a site for iodination (forming¹²⁵I-labeled peptide), which allows the coupling reaction to bequantitatively analyzed.

All peptides were synthesized at the University of Michigan Peptide CoreFacility, and the starting alginate (unmodified) was purchased fromProNova. Covalent peptide grafting onto the alginate polymer backbone isdone in a 1% (w/v) alginate solution in a 0.1 M2-(N-morpholino)-ethanesulfonic acid (MES) buffer containing 0.5 M NaCl.N-hydroxysulfosuccinimide (Sulfo-NHS) is used as a co-reactant greatlyincreasing EDC efficiencies in a similar manner to HOBt. Sulfo-NHS isadded to the reaction solution followed by the peptide and the EDC. Theratio of uronic acid:EDC:sulfo-NHS is constant, while only the peptideavailable for reaction is varied. This chemistry consistently gives65-75% coupling efficiency compared to available peptide as shown inFIG. 2. The solution is allowed to react for 14-18 hours at which timehydroxyl amine is added to quench any unreacted activatedsulfo-NHS-esters and reestablishing carboxylates. The solution isextensively dialyzed against water in 3500 MWCO dialysis tubing.Preliminary experiments utilizing ¹²⁵I-labeled GRGDY indicate <0.5% ofunreacted peptide remains after dialysis, suggesting a relatively purealginate-peptide product. The dialyzed solution is sterile filtered,lyophilized and stored dry until use. A recently described technique(Klock et al., Appl. Microbiol. Biotechnol. 40:638-643, 1994) can beused to detect any polyphenol contaminants in the alginate.

Surface modification only of alginate hydrogels will be done fortwo-dimensional cell culture experiments to save on reagents. Thisprocess is done under sterile conditions with all reactants in sterilefiltered aqueous solutions. The reaction may be done in the above MESbuffer or in diH₂O with subsequent 10-fold loss of reaction efficiency.¹²⁵I experiments show similar reaction efficiencies to the bulk modifiedalginate. Cross-linked alginate gels are cast between parallel glassplates with 2mm spacers and disks are punched out with circular punches.Ten to twelve disks are added to 50 ml centrifuge tubes with 40 mlsreaction solution with the reactants at the same ratios as above. Thedisks are extensively washed in water, and then DMEM before being placedin 24-well plates for cell experiments.

The reaction conditions have been optirized by variation of the reactionbuffer, pH, EDC:uronic acid ratio, and efficiencies of peptideincorporation between 65 and 75% are typically obtained. The density ofcell adhesion ligands, a critical regulator of cellular phenotypefollowing adhesion to a biomaterial can be readily varied over a 5-orderof magnitude density range. Both surface coupling, as well as bulkcoupling of alginate can be readily obtained with this approach.

Example 3

The adhesion of 3T3 fibroblasts and skeletal myoblasts to alginatematrices has been confirmed in culture. See FIG. 3, while no celladhesion is noted on control alginate surfaces without adhesion ligands,even in serum containing medium. Furthermore, skeletal myoblasts exhibita differentiated phenotype on these matrices. Since unmodified alginatehydrogel surfaces do not support cell adhesion, this data suggests thatinsoluble ECM signaling for cell differentiation can be partiallyprovided through coupling of cell adhesion ligands.

Example 4

The following scheme demonstrates one method of preparation of theinventive polymeric backbone materials:

This scheme displays an example of the synthetic pathways that can beused for the synthesis of the graft copolymers. Oligo-guluronate wasprepared by partial hydrolysis of alginate with 70% guluronate content.Hydrolysis occurred preferentially at the alternating region (M/Gregion), therefore, a mixture of oligo-mannuronate and oligo-guluronateresulted. The latter was separated from the other oligomer bydifferential solubility at highly acidic conditions. See Penman A,Sanderson G R (1972): A method for the determination of uronic acidsequence in alginates. Carbohydr Res 25:273:282 and Haug A, Larson B,Smisrod O (1966): A study of the constitution of alginic acid by partialacid hydrolysis. Acta Chem Scand 20:183-190. PVA, (poly(vinyl alcohol))was coupled to Boc-glycine in the presence of dicyclohexyl carbodiimide.The amount of Boc-glycine in the reaction mixture controls the branchingratio of the resulting graft copolymers in later steps. The Bocprotecting group was removed under acidic conditions and subsequentgrafting of oligo-guluronate by reductive amination of the reducing endof carbohydrate with the amino group of glycine on PVA furnished thedesired copolymers.

Example 5 Backbone: Poly(allylamine) PAM Hydrochloride

Molecular formula of repeating unit: C3H8NCl

Molecular weight of repeating unit: 93.5

Molecular weight (Mn) reported by Aldrich: 50,000-65,000

i.e., # of repeating unit on each polymer molecule: 535-695

Nondegradable from of backbone.

Side Chain: Oligo-guluronate(Gul)

Each of the sodium guluronate units has 198 MW. so 25 units has MW of4950, i.e., −5000

Comb Polymers PAM-Gul by Direct Linking of Backbone to Side Chains

Incorporation of the side chains was controlled by the ratio of Gul toPAM so as to provide polymers with 100%, 50% and 10% of sites onbackbone having side chain.

Example 6

Example 7

Polyethylene glycol (PEO) backbone (see scheme of Example 8) withbranched linker group to provide dendritic polymers when polysaccharideside chains are added. The dendritic polymers may form networks bycoordination of calcium ions between side chains of two or morediffering dendritic polymers. The linker group is hydrolyzable, and thusdegradable.

Example 8

Dendritic polylysines as the polymeric backbone for incorporation ofG-block alginate chains where prepared as shown in the followingschematic. The amino groups of lysine was protected by Boc group whilethe carboxyl end was unprotected for peptide coupling which was achievedby DCC/HOBt chemistry. PEO (8000 Mw) was first coupled tohydroxysuccinamide ester of di-Boc protected lysine in dichloromethane.The ether-washed polymer was dissolved into 25% TFA in CH₂Cl₂ to removeBoc group. Precipitation into ether furnished the deprotected peptideready for next cycle of coupling. Coupling of the corresponding freepolyamines on the polymer support with excess DCC/HOBt-activateddi-Boc-lysine furnished PEO-linked G-1, G-2 and G-3 dendrimers,respectively, after crystallization from ether. Cleavage of the peptidesfrom the polymer supports was achieved by treating the PEO-peptideconjugate in methanol with hydrazine for 1 hour producing the hydrazonepeptide dendrimers in good yields. In addition, G-2 dendrimer with afree hydroxyl end was also prepared by treating with aqueous sodiumhydroxide solution in methanol for 1 hour. PEO-Gn (n=0-3) gavesatisfactory proton and carbon NMR spectra. Purity and structures of G-2and G-3 dendrimers were established by TLC, elemental analysis and¹³C-NMR spectroscopy.

In summary, a G-3 dendritic polylysine (15 L-lysine units) of molecularweight 3096 was synthesized rapidly in 7 steps. We have designed thesedendrimers to couple G-block chains via their hemiacetal terminus. Thiswas accomplished by reductive amination using sodium cyanoborohydride.

Synthesis of PEO-Lysine dendrimer. a) TFA, CH₂Cl₂ b) L-lysine, DCC,HOBT, CH₂Cl₂.

Example 9

Comb polymers were prepared using poly(vinyl alcohol) (P)VA). PVAbelongs to a class of water-soluble polymers whose properties can bevaried widely. PVA cannot be synthesized directly due to the instabilityof its monomer. Deacetylation of poly(vinyl acetate) throughalcoholysis, hydrolysis or aminolysis leads to PVA's. The hydrophilicityand water solubility can be readily controlled by the extent ofhydrolysis and molecular weight of the poly(vinyl acetate) used. PVA isnot truly biodegradable, due to a lack of labile bonds, but PVA with amolecular weight <20K can be cleared through the kidneys and has beenused as drug delivery matrices and surgical prosthesis.

PVA (MW=9000-10000, 80% hydrolyzed) can be esterified in DMF withBoc-Glycine using the DCC coupling method, shown in the followingschematic. By varying the ratio of hydroxyl groups in the PVA to theamount of carboxylic groups in the Boc-glycine different degrees ofgrafting (i.e. 0.8, 0.25 and 0.16) can be obtained. The amino-protectinggroup (Boc) can be removed by utilization of trifluoroacetic acid inCH₂Cl₂. The polymer is characterized by standard laboratory methods suchas TLC, FT-IR, ¹H-NMR and aqueous SEC. In a second reaction step theamino functionalized PVA can be covalently coupled through reductiveamination to oligoguluronate.

Esterification of PVA 1 with Boc-Glycine 2 in DMF and subsequentdeprotection with trifluoroacetic acid in CH₂Cl₂.

Example 10

Preparation 1—Five stock solutions of aqueous sodium alginates (2%) wereprepared in Erlenmeyer flasks. Lysine ethyl ester was added to eachsolution to yield the following ratios: 0, 25, 50, 100, 150%lysine:uronic acid mole ratio. EDC and HOBT in twice the amount of molesof lysine each were added to each solution. The mixtures were thoroughlymixed and poured over petri dishes. Calcium sulfate powder was thenadded to gel the solutions for 24-48 hours. Circular discs were madefrom each batch, washed with distilled water, and lyophilized. Thedimensions and weights of each disc was measured before and afterlyophilization.

Preparation 2—Several stock solutions of aqueous sodium alginates (2%)were prepared in Erlenmeyer flasks. The selected amount of lysine ethylester (0, 25, 50, 100, 150% lysineuronic acid mole ratio) was added toeach solution and stirred for 24 hours. Each solution was poured into apetri dish to form a layer 3 mm in diameter. Calcium sulfate powder wasthen added to the surface of the layers to induce gelling. After thegels hardened (24-48 hr.), circular disks were cut from all the gels.Each set of discs were transferred into a test tube. EDC and HOBT werethen added (lysine:EDC:HOBT 1:2:2 mole ratio) to each tube. The diskswere shaken for 24 hours and rinsed with distilled water. The dimensionsand wet-weight of each disk were recorded. Each disc was then frozen,and lyophilized, and the dimensions and dry-weight of each disk wererecorded afterwards.

Study 1: Alginate gels prepared using various combinations of reagents(see Table A) using preparation 2, are tested for their ability tomaintain their structure following chelation of calcium by exposing to asolution of sodium citrate. Control alginate gels (non-crosslinked)dissolved as expected. Protected t-boc lysine was utilized as a controlin this study as the amino groups in the lysine are protected and cannotcouple to the carboxylic acid groups of the alginate. Alginate gelscross-linked with lysine using EDC alone did not dissolve, but didexpand in size following calcium chelation. Alginate gels cross-linkedusing EDC and HOBT maintained their original dimensions. These resultsconfirm that cross-linking of alginate gels leads to matrices in whichthe structure can be maintained independently of divalent cationcross-linking, and also suggest that the presence of HOBT stabilizerimproves the cross-linking.

TABLE A Gel Components Result following calcium chelation Alginate +lysine dissolved Alginate + lysine + EDC swelled in size, but did notdissolve Alginate + lysine + EDC + HOBT maintained size and shapeAlginate dissolved Alginate + t-boc lysine + EDC dissolves

Study 2: Alginate gels were cross-linked by method 2 using variousratios of alginate to lysine in order to determine if there was a dosedependency of gel stability on lysine content. Gels cross-linked withEDC alone (no HOBT) and EDC+HOBT were exposed to sodium citrate andexamined for swelling or dissolution. Gel dissolution and stability wasa function of lysine content and cross-linking conditions as shown inTable B.

TABLE B Lysine content (% alginate Cross-linked with functional groups)Cross-linked with EDC alone EDC + HOBT 0 dissolved dissolved 25dissolved dissolved 50 did not dissolve, but swelled mild swelling todimensions of container 75 mild sweillng maintained size and shape 100mild swelling maintained size and shape 150 small amount of swellingmaintained size and shape

Study 3: Alginate gels cross-linked with lysine (100% lysine content,cross-linking with EDC and HOBT) were cut into slabs (initial volume 576mm³), lyophilized and then tested for their shape memory (see FIG. 4).The dried matrices (right side of figure) were compressed and placed intubing (middle of the figure) with an inner diameter of 1.56 mm(approximately 4.5 French—a tubing diameter typically utilized forendoscopic procedures), and pushed through a 5 cm long portion of thetubing. The matrices were then placed in a petri dish containing waterand observed over time. These matrices returned to a slab geometry (leftside of the figure) after 1 hour, and obtained a volume of approximately400 mm³ (approximately 70% initial volume) by this time. The ability ofthese matrices to return to their approximate size ad shape after thissevere compression indicates they have significant shape memory.

Study 4: An important feature of the cross-linked matrices is theirability to be seeded with cells after preparation. Cross-linked alginategels (100% lysine content, EDC+HOBT to cross-link) were lyophilized andsterilized. Scanning electron microscopy examination of matricesrevealed a highly porous material with large pores (FIG. 5). Thematrices were placed in a suspension of smooth muscle cells in tissueculture medium (DMEM supplemented with 10% calf serum), and examined forcell infiltration. Observation of matrices with a microscope indicatedthat the cell suspension absorbed into the cross-linked alginatematrices, and this resulted in a distribution of cells throughout thematrices (not shown in figure).

Study 5: 2% alginate gels were crosslinked with lysine (100% lysine)using method one, however, 10-fold EDC and 5-fold HOBT concentrationswere used to optimize crosslinking. 20 g (approximately 20 mls) of 2%alginate solution were added to 50 ml conicals and selected lysine (0%,1%, 10%, 25%, 50% and 100% lysine compared to alginate monomer units)and HOBT amounts were added. The solutions were mixed well, thenappropriate amounts of EDC were added. Immediately following theaddition of EDC, 0.168 grams calcium sulfate was added and the conicalswere shaken vigorously for 10 to 20 seconds prior to pouring thesolution. The gels were cured for 3 hours between parallel plates about2.5 mm apart. Disks were then punched out and added to either sodiumcitrate (to remove the calcium) or 0.1 M calcium chloride for 10 minutesprior to storage in distilled water. Gel stability with removal ofcalcium and mechanical testing were done with all conditions.

removal of compressional compressional calcium with modulus (gels withmodulus (calcium % Lysine sodium citrate calcium) removed)  0% dissolved 6.4 N/mm dissolved  1% dissolved  4.6 N/mm dissolved 10% dissolved 3.58N/mm dissolved 25% mild swelling 1.04 N/mm 0.143 N/mm 50% littleswelling 0.55 N/mm 0.174 N/mm 100%  little swelling 0.32 N/mm  2.02 N/mm

The dose dependency of the lysine content from study 2 was reconfirmed,but the extent of crosslinking was increased as suggested by completestability of the 25% and greater crosslinked gels upon calcium removalThe compressional modulus of the gel disks decreased with increasinglysine content likely due to the lysine disruption of the calciumbinding sites in the alginate. Interestingly, upon calcium removal withsodium citrate, the modulus of the gel disks increase with increasinglysine content.

Example 11

Alginate cross-linking with diamines is performed in a 0.1 M MES bufferof pH 7 with 0.3 M NaCl. The chemistry has been optimized for maximumcross-linking with the variables pII, [NaCl], and EDC:HOBt:uronic acidratio. EDC reacts with the carboxyl group of the uronic acid creating anactivated ester intermediate which is reactive towards amines. A majorcompeting reaction with amide bond formation is hydrolysis of the EDCintermediate by water, and the EDC intermediate half-life is on theorder of seconds (Hermanson, 1996, cited above). However, the additionof coreactants like HOBt or N-hydroxysulfosuccinimide will react withthe EDC activated ester creating longer lasting intermediates leading togreater reaction efficiencies (Hermanson, 1996, cited above).

Example 12

The reaction scheme for PEG cross-linking molecules, shown belowinvolves adding equimolar amounts based on alcohol groups ofpoly(ethylene glycol) (1) and N-protected amino acid (2), andesterifying via direct coupling by DCC and DMAP in CH₂Cl₂. Deprotectionof the BOC protecting group in compound (3) is performed by utilizationof trifluoroacetic acid (TFA). Cross-linking reactions between thecarboxylic group of the alginate and the primary amino groups of themodified PEG molecule are carried out by the formation of an hydroxybenzotriazole active ester in situ and the consequent addition of thecoupling agent EDC. The functionalized PEG's were purified by liquidcolumn chromatography and characterized utilizing standard laboratorymethods such as TLC, FT-IR, ¹H-NMR and elemental analysis. The molecularweight distribution as well as structural information of the polymerswill be determined by GPC measurements in aqueous solution. We areutilizing a relatively new analytical technology known as SEC³ (RI,Viscometer, RALLS) to obtain absolute molecular weights thus eliminatingthe assumption that standard and sample have the same molecularstructure.

Poly(ethylene glycol) with molecular weights MW 200, 400, 600, and 1000was obtained from Lancaster Synthesis Inc., PEG with MW 3400 and1,3-Dicyclohexylcarbodiimide (DCC) were from Aldrich chemical company.N-t-boc-glycine (98%), trifluoracetic acid and1-ethyl-3-[3-dimethylamino-propyl]carbodiimide (EDC) were ordered fromSigma, St. Louis, Mo.

(A) Synthesis of amino terminated Poly(ethylene glycol)s with variousrepeat units n ranging from 4.5-77.3 (MW=200, 400, 600, 1000, 3400).

(B) Reaction of PEG-cross-linker molecule with sodium alginate.

The reaction solutions are cast between parallel glass plates for 12-16hours and defined shapes may be cut from these hydrogel sheets. Definedshapes may also be formed by casting the reacting solution into a moldof the desired shape, and allowing the cross-linking reaction to occur.The resultant hydrogels are characterized for mechanical properties(elastic modulus, shear modulus, maximum true stress, maximumextension), and swelling properties. The methyl ester of lysine andmodified amino terminated PEG of molecular weights 200, 400, 600, 1000and 3400 was used to cross-link alginate in ratios of amino:carboxylicgroups 3-50%.

Example 13

Lysine cross-linked alginates. The alginate hydrogels were cross-linkedwith the methyl ester of lysine. The carbodiimide chemistry wasoptimized for maximum effective cross-linking at any given lysinecontent by varying pH, ionic strength of the reaction buffer, and theEDC:HOBt:uronic acid ratio. The mechanics of the hydrogel network can becontrolled by varying the amount of lysine available for cross-linking(FIG. 6). The elastic modulus of the hydrogels increases with increasinglysine content up to 35%, but then decrease with additional lysineadded. This decrease in modulus is attributed to an increase in networkdefects including more dangling half-reacted lysines and elasticallyineffective loops which do not contribute to the mechanical propertiesof the network, and in this case actually detract from the mechanics dueto the shear number of defects.

The mechanical properties, including stiffness shown by the elasticmodulus, as well as strength and elasticity, can be controlled byvarying the amount of lysine available for reaction.

The swelling capabilities of the lysine crosslinked hydrogels weredetermined by measuring the volume of water a known mass of crosslinkedalginate absorbs. Swollen hydrogel disks were cut to 15.7 mm indiameter, dried with a towel, and weighed to determine the weight of thewater and polymer. The disks were then frozen and lyophilized to removeall of the water from the hydrogels, leaving highly porous fluffy disksafter lyophilization. The initial wet weight of the hydrogels wasdivided by the dry weight of the dried disks to determine the swellingratio (FIG. 7). Swelling ability of lysine crosslinked alginatehydrogels decreases with increased crosslinking. Lightly cross-linkedalginates absorb over 2000× their mass in water, suggesting they will beuseful as superabsorbent materials (e.g., in disposable diapers). Themore highly crosslinked gels absorbed approximately 70 times theirweight in water, with intermediate crosslink densities ranging betweenthese two extremes. The dry disks were then rewet with diH₂O over 48hours and weighed to determine if the lyophilized disks were able toabsorb comparable amounts of water that they initially held. Thelyophilized disks were able to absorb similar amounts of water that theyinitially held +/− about 10%. The more highly crosslinked gels (75%lysine) absorbed approximately 10% less water and the lightlycrosslinked gels (1-20% lysine) absorbed 10% more than initiallymeasured. On drying, the alginate network phase separates to make ahighly porous sponge. The hydrated network microstructure does not seemto reform immediately upon rehydration, as much of the water soaked upby the sponges may be removed by placing the sponge on a towel withinthe first few hours after rehydration. However, after 48 hours ofsoaking very little water may be removed from the from the structurewhen exposed to a drying towel suggesting the hydrated network structurereturns.

Example 14

Highly cross-linked alginate matrices exhibit shape memory propertiesadvantageous for certain applications. Lyophilized alginate matrices actas hydrophilic sponges when exposed to water, hydrating almostinstantly. To demonstrate shape memory properties, 50% lysinecrosslinked sponges were compressed, rolled up into tight cylinders anddelivered through 3mm ID silicone tubing to mimic endoscopic delivery onthe laboratory bench. The rolled up matrices were pushed through thetubing with flexible teflon string (1.3 mm OD), and they returned totheir initial shape and dimensions within seconds upon rehydration. Thewater absorption properties of the compressed disks are similar tononcompressed disks (in the above swelling study) and absorb around 90%of the initial water content after 24 hours.

Example 15

The molecular weight between cross-links in the alginate network wascalculated directly from the shear modulus and swelling measurements(Peppas and Merrill, J. Appl. Polym. Sci. 21: 1763-1770, 1977) to assessthe density of functional cross-links (interchain). This number can thenbe compared to the total number of cross-links (intra- and interchain)determined chemically. Calculation of M_(c) was performed utilizing therelationship:

G=RTC _(r) /M _(c)(1−2M _(c) /M _(n))Q ^(−⅓)  Equation 1

where G is the shear modulus, R and T are the gas constant andtemperature in Kelvin respectfully, C_(r) is the concentration of thepolymer in the cross-linking solution, M_(c) is the molecular weightbetween cross-links, and M_(n) is the number average molecular weight ofthe native polymer. Q is the swelling ratio defined as

Q=v _(f) /v _(s)  Equation 2

with v_(r) being the volume fraction of the polymer in the unswollencross-linked gel and v_(s) is the volume fraction in the swollen gel. Gis obtained from manipulation of the stress-strain data from compressiontesting by plotting stress versus −(λ_−_(—)1/λ²) with

λ=L/L ₀  Equation 3

L₀ and L being the thickness of the gel before and during compressionrespectfully.

These calculations indicate that Mc ranges from 1500 for the most highlylysine cross-linked alginates, to approximately 25,000 for the leastcross-linked (1% lysine) alginates.

Example 16

Studies have also been performed to vary the Mc by altering themolecular weight of the cross-linking molecule in polyethylene glycolcross-linked alginates. These studies have utilized PEG diaminessynthesized in our laboratories as the cross-linking molecule. Thecompressive modulus of alginate gels increased with the number of repeatunits in the cross-linking molecule up to a molecular weight of 1000.See FIG. 8. Showing the elastic modulus in compression [KPa] versusnumber of repeat units in cross-linking molecule. An equal molar amountof each monomer was used in each reaction. This may be related todirectly to the molecular weight of the cross-linking PEG, or to anincreased flexibility of the cross-linking monomer which results in agreater extent of reaction (Allen et al, Macromolecules, 22:809-816,1989). A decrease in the modulus was found with further increase in thecross-linking molecule molecular weight. Varying the cross-link density(utilizing the PEG with 1000 molecular weight), again had a strongeffect on the stiffness of the gels. Similar to the results with lysine,an increase in modulus was noted up to a certain cross-linking, but thendecreased with additional cross-linking. FIG. 9 showing elastic modulusvs cross-link density [%] utilizing the PEG of 1000 molecular weight asthe cross-linking molecule. This decrease in modulus is again likelyattributable to an increase in network defects at higher PEG diamineconcentrations, including more dangling half-reacted PEGs which detractfrom the mechanics.

Example 17

Cross-linked polyaldehyde alginates (PAA). An additional approach tocovalently cross-link alginates is to oxidize alginate and cross-link itwith a bifunctional cross-linker to form hydrogels. Thus, alginate, 1,was derivatized by sodium periodate oxidation, as shown below, atambient temperatures to yield the limit-oxidized product, 2. Thereaction was monitored by the appearance of the aldehydic symmetricvibrational band (carbonyl) via FTIR. Limit-oxidized alginate was thencross-linked via the aldehyde groups with adipic dihydrazide to formhydrogels, 3. This process was followed by the disappearance of thesymmetric vibrational band at 1735 cm⁻¹.

Further, limit-oxidized alginates were cross-linked with adipicdihydrazide at various % w/w alginates. The compressive modulus of theresulting gels was measured and evaluated (FIG. 10). Gelling was set at3% w/w alginates with a modulus of 200 kPa, and increased with thealginate percentage to reach 900 kPa at 10% w/w alginate content. Thiscross-linking procedure provides a wide range of control (700 kPa) overthe mechanical strength of alginate-based biomaterials.

The mechanical strength of cross-linked limit-oxidized alginates alsodepended on the concentration of the cross-linker as well as the calciumion content in the final gel. The compressive modulus increased with theconcentration of adipic dihydrazide in the gel (FIG. 11). For example,at 25 mM adipic dihydrazide, the modulus was at 200 kPa and increased to100 kPa at 150 mM. No difference was observed at higher concentration ofthe cross-linker. A significant increase of 250 kPa was also observedfor the compressive modulus as the calcium ion concentration increasedfrom 10 mM to 30 mM (FIG. 12).

Example 18

The polyguluronate sequence responsible for alginate gelation wasisolated, derivatized and cross-linked to form hydrogels, analogous tothe scheme shown in Example 17, but for an un-crosslinked material.Thus, sodium polyguluronate, 1, was isolated from alginates by acidhydrolysis following a modified procedure (Haug, A.; Larsen, B.;Smidsrod, O. Acta Chem. Scand. 1966, 20, 183-190; and Haug, A.; Larsen,B.; Smidsrod, O. Acta Chem. Scand. 1967, 21, 691-704). The product wascharacterized by FTIR, H-NMR, and ¹³C-NMR and correlated well with thereported characterizations (see also Penman, A.; Sanderson, G. R.Carbohyd. Res. 1972, 25, 273-282). Sodium polyguluronate was derivatizedby sodium periodate oxidation at ambient temperatures to yieldpolyaldehyde guluronate (PAG), 2. The degree of oxidation was controlledby the mole equivalent periodate used in each reaction. The reaction wasmonitored by the appearance of the aldehydic symmetric vibrational band(carbonyl) via FTIR. PAG was then cross-linked via the aldehyde groupswith adipic dihydrazide to form hydrogels, 3. This process was followedby the disappearance of the symmetric vibrational band at 1735 cm⁻¹.

A common approach for the immobilization of molecular probes andproteins onto proteoglycans is the partial periodate oxidation thepolysaccharide portion of the proteoglycan followed by coupling via theformation of a Schiff or hydrazone linkage. The same basic approach wasutilized to couple a bifunctional cross-linker to partially oxidizedsodium polyguluronates. This cross-linking provides an additional levelof control, beside ionic cross-linking, over the mechanical stabilityand strength of the hydrogel under investigation.

Example 19

To achieve an understanding over the factors behind the gellingproperties of certain materials, it was essential to investigate theeffect of varying the concentration of polyaldehyde guluronate, adipicdihydrazide, and calcium ions in the resulting gels. Hence, gels werecross-linked at various concentrations of polyaldehyde guluronate andthe compressive modulus was measured and plotted against final % w/w PAG(See FIG. 13). Whereas no hydrogels formed with 4% w/w PAG and below,even after 48 hours time interval, cross-linked polyaldehyde guluronategelled starting at 5% w/w PAG with a compressive modulus of 82 kPa. Thecompressive modulus then increased as the PAG content in the final gelincreased to reach 880 kPa at 10% w/w PAG. This was expected since thenumber of aldehydic functional groups increased with the PAG content inthe gel. Hence, the efficiency of the cross-linker increases and resultsin a larger modulus. As a result, varying the % w/w PAG in the finalgel, can provide a control over the elasticity as well as the strengthof the corresponding gel.

The mechanical strength of gels can be increased by increasing thedegree of cross-linking. Hence, 6% w/w PAG was cross-linked at differentconcentrations of adipic dihydrazide and the compressive modulus wasevaluated. It was found that increasing the concentration of adipicdihydrazide resulted in an increase in the compressive modulus of 6% w/wPAG. An optimal value of 560 kPa was obtained for the modulus at aconcentration of 150 mM adipic dihydrazide. As the concentration ofadipic dihydrazide was increased further, the modulus decreased to 350kPa (See FIG. 14). Theoretically, the efficiency of cross-linking shoulddecrease when the amount of hydrazide functional groups exceed thenumber of aldehydes in the polymer. In other words, at high adipicdihydrazide concentrations, the cross-linker reacts with only onealdehydic group while the other terminus does not react. As a result,the degree of functional cross-linking will decrease even though thatthe degree of incorporation of the adipic dihydrazide have alsoincreased.

In comparison to unmodified alginates, cross-linked polyaldehydeguluronates can also be controlled by the amount of calcium ions presentin the these materials. PAG (6% w/w) was cross-linked with adipicdihydrazide (150 mM) at various concentrations of calcium and sodiumchloride (such as 10, 20, 40, 80, and 100 mM). The compressive modulusof the resulting gels increased with increasing calcium concentrationsto an optimal value of 600 kPa at 40 mM calcium chloride (See FIG. 15),wherein the open block, ▭, is sodium chloride and the closed block, ,is calcium chloride). Above this concentration, there was no statisticaldifferences in the compressive modulus. This indicates that ioniccross-linking, similar to that in alginates, could provide another levelof control over the mechanical properties of these materials. Toeliminate the contribution of the ionic strength to the increase in thecompressive modulus, PAG was also cross-linked in the presence of sodiumchloride at the same concentrations as above. Even though a slightincrease in the modulus was observed initially as sodium ions contentincreased, the value of the modulus leveled out at 390 kPa. There was asignificant difference of 210 kPa between the optimal modulus in thepresence of calcium and sodium ions. This difference in the compressivemodulus (150 kPa) clearly demonstrates that the presence of calciumindeed contributes to the mechanical strength of these materials.

One potential application for these materials is their use as threedimensional matrices for cell transplantation as mentioned above. Hence,to ensure cell survival and proliferation in these materials, it wasnecessary to investigate their gelling behavior at physiologicalconditions (pH 7.4). By adjusting the pH of these materials to 7.4, aslight decrease in the compressive modulus was observed. For example, ata pH of 7.4, no gel was formed at 5% w/w PAG, whereas at lower pHgelling was set starting with 5% w/w PAG (See FIG. 16), wherein the openblock, ▭, is at pH 7.4 and the closed block, , is at pH<7). Moreover,the modulus was 600 kPa at 10 % w/w PAG compared to 880 kPa for theoriginal gel condition. This was expected since it is well known thatthe reactivity of hydrazide groups with aldehydes is optimal at lowerpHs. Under acidic conditions, aldehydes are protonated and, hence, aremore susceptible to nucleophilic attack by the hydrazide groups. Atneutral to basic conditions however, slower kinetics are in effect and alonger time interval is required for the completion of the reaction.This results in a lower degree of cross-linking which directly causes adecrease in the compressive modulus.

The degree of cross-linking in these materials can also be controlled byvarying the degree of oxidation of the polyguluronate chains (seePainter, T.; Larsen, B. Carbohyd. Res. 1969, 10, 186-187; Painter, T.;Larsen, B. Acta Chem. Scand. 1970, 24, 813-833; and, Ishak, M. F.;Painter, T. Acta Chem. Scand. 1971, 25, 3875-3877).

This provided another control over the number of aldehydic units on thepolyguluronate strand that are available for cross-linking. As a result,polyguluronate was oxidized using various amounts of sodium periodateand cross-linked at 10% w/w PAG with adipic dihydrazide at the optimalconcentration of 150 mM in 24 well plates. All materials gelled,starting at 20% theoretical oxidation with a compressive modulus of 500kPa (See FIG. 17). The modulus increased with the percentage ofoxidation of polyguluronate to reach a maximum value of 1000 kPa at 100%theoretical oxidation.

These results clearly indicate that a wide range of mechanical stabilitycould be achieved by varying the degree of oxidation of polyguluronates.Whereas cross-linked 20% oxidized PAG exhibited weak gels comparablewith alginates, 80% oxidized PAG and above were stiff and brittle withhigh compressive moduli. These characteristics are very desirable inprocessing biomaterials for cell immobilization and as drug deliverysystems as well. Depending on the degree of oxidation of the polymer,devices with specific pore sizes and mechanical strength can be providedto deliver cells or therapeutic drugs to the site of implantation.

Example 20

A critical question, in terms of cell transplantation, is whether cellspresent in the un-crosslinked monomers will be harmed by thecross-linking reaction. Smooth muscle cells (rat aorta-derived) wereplaced into a PAG solution, and then cross-linking via addition ofadipic acid dihydrazide. Incorporation of the smooth muscle cells withinthe gels was approximately 100%, and the cell number and metabolicactivity of the cells was maintained for the 7 days of the experiment.These results indicate that cells can be transplanted in materialscross-linked with this chemistry (PAA polymers also utilize samecross-linking). We did not expect cells to proliferate in thesematrices, as they do not contain cell adhesion ligands, and we did notobserve any proliferation. RGD-containing and other cell adhesionpeptides can be readily coupled to these polymers as described above toenhance the cell interaction. Coupling efficiencies of approximately 70%have been achieved using the coupling chemistry optimized for alginate.

Example 21

The suitability of these polymers as delivery vehicles for angiogenicfactors was also investigated. VEGF was mixed with the PAG, and then thePAG was cross-linked. VEGF release was monitored by adding trace amountsof ¹²⁵I-VEGF. PAG (20% w/w ) was cross-linked with adipic dihydrazidecontaining ¹²⁵I-labelled VEGF in the presence of calcium chloride, andmixture was allowed to gel for one hour and then incubated at 37° C. inDMEM medium. The medium was replaced with fresh medium and counted forits radioactivity. Little to no burst release of the VEGF was observedin any of the experimental conditions (See FIG. 18), wherein the openblock, ▭, is with heparin and the closed block, , is without heparin).In the presence of heparin, the VEGF was released at a rate of 7% totalincorporated growth factor/day for 5 days, then a slower release of2%/day for the next 14 days. In the absence of heparin, a higher initialrelease of 9%/day for 5 days was observed, followed again by a slowerrelease of 2%/day for the next 14 days.

Example 22

Polyaldehyde guluronate was also cross-linked with adipic dihydrazide inthe absence of calcium chloride at 10% w/w PAG. A slower release of¹²⁵I-VEGF from these materials was observed in both cases, with and without heparin. In the presence of heparin, VEGF was initially released ata rate of 4%/day for 5 days, followed by a slower release at a rate of0.2%/day for 14 days (See FIG. 19), wherein the open block, ▭, is withheparin and the closed block, , is without heparin). In the absence ofheparin, VEGF was released at a rate of 6%/day for five days, followedby 0.8%/day for the next 14 days. These experiments suggest that it ispossible to control the rate at which VEGF is released from thesematerials by controlling the presence of calcium and heparin. Weanticipate that the release will be strongly dependent on the PAGconcentration and cross-link density as these variables will regulatethe pore size in the hydrogel. It is possible to achieve release ratesover a very wide range by altering these variables.

Example 23

A cell adhesion ligand, GRGDY, was coupled to sodium poly(guluronate)and PAG with the same type of EDC chemistry utilized for coupling toalginate. To monitor the degree of coupling, trace ¹²⁵I-GRGDY was mixedwith the adhesion peptide and the mixture was dialyzed against doubledistilled water. The dialyzate was counted in a liquid scintillationcounter to determine the amount of radioactive material present. In theabsence of EDC, only 1.5% GRGDY was present in sodium poly(puluronate),whereas, in the presence of EDC, 61% peptide was incorporated (See FIG.20). In PAG material, 55% GRGDY was incorporated with EDC compared to24% in the absence of EDC.

Different chemistry can also be used to couple this ligand to PAG. Thisapproach utilizes the reductive amination coupling of the aminefunctional groups on the terminus of the peptide and the aldehydic groupabundant in the PAG material. Essentially, the amine reacted with thealdehyde to form a labile imine bond which was reduced using sodiumcyanoborohydride (NaCNBH₃) to form a stable amine linkage. The degree ofincorporation of the peptide into the PAG material was 65%. The iminebond between the amino terminal of the peptide and the aldehyde group ofPAG formed with this reaction is likely also forming when the EDCchemistry is utilized to couple the peptide. This reaction ties up someof the peptides and prevents them from reacting with the activatedcarboxylic acid groups, hence, resulting in a lower degree ofincorporation of the peptide (55%). The same reaction essentiallyexplains the reason behind the incorporation of the peptide in theabsence of any additive (24%).

This new coupling chemistry can also be used to chemically bind otherpeptides and proteins (e.g., growth factors) or drugs to PAG andlimit-oxidized alginates. Importantly, this reaction results in theformation of a labile bond that will degrade and release the boundmolecule. This will allow drugs to be released slowly from the matrices,and this release will be chemically controlled, diffusion controlled orcontrolled by both processes. This bond can be easily reduced usingsodium cyanoborohydride to yield a very stable bond if one wishes thebound molecule to remain bound (e.g., cell adhesion ligand). Any growthfactor having pendant amino groups can be coupled with this reaction.Pharmaceutical drugs that could potentially be used will have aminogroups available from the imine bond formation according to the schemebelow for example. In addition to the amine group, growth hormones anddrugs could be modified to incorporate free hydrazines, hydrazides, orsemicarbazides groups that can form hydrazone or semicarbazone linkagesrespectively. This will allow for a different release of the drug orhormone and consequently provide another level of control over the rateof release.

Incorporation of VEGF in limit oxidized alginates and PAG matrices

Example 24

PEG hydrazide cross-linkers. PAG can be crosslinked with adipic aciddihydrazide. Dihydrazide cross-linkers can be synthesized with variouslengths starting with a polyethylene glycol core. Poly(ethylene glycol),PEG, with molecular weights of 200, 400, 1000, and 3400 can be reactedwith succinic anhydride in the presence of N,N-dimethylamino pyridine toform poly(ethylene glycol disuccinate)

The ester bonds formed between the PEG core and the succinate groups arebiodegradable. Hydrazine, can then be coupled to the terminals of thesepolymers using DCC chemistry to yield polyethylene glycol dihydrazides.By starting with PEGs with different molecular weights, dihydrazidecross-linkers with various chain lengths can be synthesized. Thesepolymers can be used to cross-link poly(aldehyde guluronates), PAG.

Example 25

To gain more control over the degradability of PAG materials, a methodto synthesize cross-linkers with nondegradable cores is provided.Reacting PEG with different molecular weights with methyl chloroacetateswill form dicarboxymethyl-PEG which will then be coupled to hydrazine atboth terminals to yield PEG dihydrazide.

These dihydrazides have a non-degradable core with ether linkages. Asbefore, controlling the molecular weights of the starting PEG polymerswill yield PEG dihydrazides with various chain lengths.

These dihydrazides can be used to cross-link PAG and form materials withonly one degradable linkage which is the hydrazone bond. This bond canbe further stabilized by borohydride reduction to yield nondegradablematerials. Hence, PAG polymers can be crosslinked with dihydrazides toform non-degradable materials, materials with hydrazone degradablebonds, or materials with hydrazone and ester bonds both of which aredegradable. This approach will provide materials with various rates ofdegradation. Moreover, by selecting the appropriate length of PEGpolymers used the mechanical properties of the resulting cross-linkedbiomaterial can be controlled.

Example 26

A photopolymerizable polyguluronate can be synthesized from hydrazidoacrylate monomers coupled to G-block polyguluronate via the aldehydicterminus. These materials can then be injected into the desired site andpolymerized photochemically to form hydrogels. Hydrazido acrylate can besynthesized starting with acryloyl chloride and t-butylcarbazate to formthe protected hydrazido acrylate.

Deprotection using trfiluoro acetic acid, TFA, will afford the desiredmonomer. This methodology provides a means to deliver G-block into thedesired site and polymerize it afterwards via photoinduced free-radicalpolymerization. Hydrazides react with aldehydes as in the case of PAG.In this example, the hydrazides react with the hemiacetal terminal ofG-block to form an acrylic hydrazone terminal on the G-block chain.Hydrazido acrylates were chosen because of the ease of incorporatingthese functional groups in G-blocks.

These materials could be prepolymerized into the desired shape and usedas three-dimensional matrices for cell transplantation, or alternativelymixed with cells and injected as solutions into the implantation site.Photoinduced free-radical polymerization of the acrylate groups wouldthen provide a non-degradable backbone.

These monomers can be copolymerized with acrylic acid, acrylamide, MMA,HEMA, HPMA, allyl amine, dimethylallyl amine, or other monomers withsimilar functionality. The degree of G-block incorporation can becontrolled by varying the percentages of co-monomer used.

Example 27

Copolymerizing G-block hydrazido acrylates with diallyldimethyl ammoniumchloride monomers and allyldimethyl ammonium chloride in aqueoussolutions using ammonium persulfate as the initiator provides G-blockincorporated polymers with various rigid backbone structures.

The pyrrolidine unit formed after the polymerization restricts themobility of the polymer due to its cyclic structure and renders thebackbone more rigid. The stiffness of the backbone is then controlled bythe percentage of diallyldimethyl ammonium chloride units incorporated.

Another approach for the synthesis of these polymers is to prepolymerizehydrazido acrylates with diallyl dimethyl ammonium chloride andallyldimethyl ammonium chloride monomers.

The polymerization is accomplished using ammonium persulfate in aqueoussolutions. Afterwards, G-block is coupled to the hydrazido groups toform degradable hydrazone linkages. The hydrazone bond can then bereduced with sodium cyanoborohydride to form the more stable hydrazidelinkages.

Example 28

A common approach to control the mechanical strength of poly acrylatesand derivatives is by cross-linking these polymers With acrylate-basedcross-linkers (Naghash et al., Polymer, 1187-1196, 1997; Dietz andPeppas, Polymer, 3767-3781, 1997). Ethyleneglycol dimethacrylate,hexaethyleneglycol dimethacrylate, and other bifunctional andmultifunctional cross-linkers can crosslink G-block-hydrazido acrylatemonomers.

Again, by controlling the percentage of cross-linker in the finalproduct. we can control the mechanical properties of the resultingpolymers.

Example 29

Dendritic polymers can be provided by coupling of G-block to PEG-lysinedendritic polymers. It is well known that the hemiacetal terminus ofmonosaccharides and polysaccharides is in equilibrium with the openaldehyde form.

Moreover, the reaction of amines with aldehydes is also in anequilibrium. In the presence of sodium cyanoborohydride, the imine bondformed between the amine arid the aldehyde is reduced which shifts theequilibrium to form more aldehydes and drive the reaction to completion.This process is sluggish, however, and a faster and more efficientmethod is needed to couple G-block to different polymers. Thus,hydrazide functional groups can be used to achieve this coupling.Hydrazines, hydrazides, and semicarbazides react with aldehydes to formimine-like bonds. This process does not depend on the presence ofborohydrides. Hydrazide moieties are more nucleophilic than amines andcan attack electrophilic centers like carbonyl groups much faster. Inaddition, sodium cyanoborohydride could eventually be used later toinduce stability on the hydrazone or semicarbazone linkage.

Example 30

Dendrimers with reactive functional groups on the terminals for G-blockcoupling can be synthesized using a similar method to the one used forlysine dendrimers and the end groups modified to semicarbazideterminals.

As before, we start with polyethylene glycol, PEG, and couple it to(Boc)2-lysine via DCC chemistry, and deprotect with trifluoro aceticacid, TFA, to form PEG dilysinate. Allowing the PEG dilysinate to reactwith excess alkyl diisocyanate will provide polymers with an isocyanategroup on the terminals. Reacting the isocyanate groups with hydrazinewill finally afford the modified dendrimer with four semicarbazidegroups available to couple G-block. The same methodology can essentiallybe used to synthesize PEG hexalysinate, PEG octalysinate, and so on.

Example 31

Comb polymers. Monosaccharides and oligosaccharides have beensuccessfully incorporated into the backbone of polyacrylamides(Calistrom and Bednarski, MRS Bulletin: 54-59, 1992). Similarmethodologies can be used to incorporate G-block into the backbone ofseveral synthetic polymers. Three methodologies are followed for thesyntheses of new biomaterials. The first is by utilizing a poly(vinylalcohol) backbone functionalized with hydrazido groups onto whichG-block chains are coupled. The second method utilizes poly(allylamine)backbones which are also modified to incorporate reactive hydrazidogroups for G-block coupling. These backbones are chosen because they arebiocompatible and are easily excreted by the kidney with molecularweights of 10,000 or less. The third approach involves couplingpolyguluronate to polyaldehyde guluronate. This will result in theformation of a polymer comprised completely of alginate derivedmolecules.

PVA-based Materials

Low molecular weight poly(vinyl alcohol), PVA, can be modified byreaction with succinic anhydride in the presence of N,N-dimethylaminopyridine, DMAP, to afford poly(vinylsuccinate) intermediate.

The ester bond thus formed between PVA and the succinate group is abiodegradable bond susceptible to enzymatic cleavages in biologicalsystems. Hydrazine, can then be coupled to this intermediate via DCCcoupling to form poly(vinylhydrazidosuccinate). Therefore, the degree ofhydrazide incorporation can be controlled in the final product bycontrolling the number of succinic anhydride molecules used in theprevious reaction. The control over the degree of hydrazideincorporation is crucial since this will dictate the degree of G-blockincorporation in the next step.

Other hydrazide groups which can be incorporated in the PVA backbone areoxalyl dihydrazide (n=0), malonic dihydrazide (n=1), succinicdihydrazide (n =2), adipic dihydrazide (n=4), suberic dihydrazide (n=6),and others. These dihydrazides will provide various lengths for thespacer arms to be incorporated between the PVA backbone and the G-blockchains.

Due to the reactivity of hydrazides toward aldehydic groups, the sameapproach can be used to attach G-block chains to this polymer via thehemiacetal terminus. This provides a synthetic backbone polymer ontowhich G-block is linked via a degradable linkage.

This linkage can be further stabilized by reduction with sodiumcyanoborohydride. The degree of G-block incorporation in the finalmaterial will exhibit a direct relationship with the gelling propertiesas well as the strength of the hydrogel formed.

Poly(allylamine)-based Materials

A similar approach is to be used for the modification ofpoly(allylamine) by reacting it with succinic anhydride followed byhydrazide incorporation using carbodiimide chemistry to formpoly(N-allylsuccinamidohydrazides).

As in the previous example, the reactive hydrazido groups provide ameans to attach G-block to the polymer backbone via the hemiacetalterminus. In contrast to the PVA-based materials, the amide bond formedbetween the poly(allylamine) and the succinate group is a non-degradablebond.

The coupling of G-blocks to both polymer backbones will formbiodegradable linkages in the form of hydrazones.

These linkages can be further reduced with sodium cyanoborohydride toform a more stable hydrazine linkage that is non-degradable. It istherefore possible to tailor biomaterials with varying rates ofdegradation depending on the synthetic methodology followed.

PA G-based Comb Polymers

Polyaldehyde guluronate, PAG, was allowed to react with hydrazine andsodium borohydride to afford the polyhydrazino guluronate derivative.The hydrazine groups on this alginate derived polymer are used toincorporate G-block chains via their hemiacetal termini.

This will provide biocompatible and biodegradable materials fromnaturally derived polysaccharides with hydrolyzable hydrazone linkages.Hydrolysis of the hydrazone linkage in these materials will lead toshort chain polysaccharides that can be excreted by the kidney. Furthermore, reduction of the hydrazone bond by borohydrides can form achemically stable hydrazine bond that provide non-degradable materials.This will again provide both biodegradable and non-degradablebiomaterials derived from natural polysaccharides.

The preceding examples can be repeated with similar success bysubstituting the generically or specifically described reactants and/oroperating conditions of this invention for those used in the precedingexamples.

From the foregoing description, one skilled in the art can easilyascertain the essential characteristics of this invention and, withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions.

What is claimed is:
 1. A modified alginate, which comprises at least onealginate chain section to which is bonded by covalent bonding at leastone cell attachment peptide or RGD peptide which promotes cell adhesionand growth.
 2. The modified alginate of claim 1, wherein the moleculeuseful for cell adhesion and growth is bonded through a uronic acidresidue on the alginate chain section.
 3. The modified alginate of claim1, wherein the alginate chain section comprises an oligomeric block unitof D-mannuronate, an oligomeric block unit of L-guluronate, anoligomeric block unit of D-mannuronate and L-guluronate or a mixture ofsaid block units.
 4. The modified alginate of claim 1, wherein thealginate chain section has a molecular weight of less than about 50,000.5. The modified alginate of claim 1, wherein the alginate chain sectionhas a molecular weight of less than about 30,000.
 6. The modifiedalginate of claim 1, wherein the alginate chain section has a molecularweight of about 100,000 or more.
 7. The modified alginate of claim 1,wherein the alginate chain section is a naturally occurring alginate. 8.A modified alpinate of claim 1, wherein the modified alginate containsat least one alginate chain sectiononded to a polymeric backbone sectionor at least one alginate chain section crosslinked to another alginatechain section on the same or a different molecule.
 9. A method for celltransplantation into a system which comprises implanting a modifiedalginate of claim 8 in a matrix form into the system and subsequentlyintroducing the cells for transplantation into the matrix.
 10. A methodfor treating a human or animal which comprises administering thereto amodified alginte of claim 8, as a matrix for bone or soft tissuereplacement.
 11. A method for drug delivery which comprisesadministering to a human or animal a modified alginate of claim 1,wherein the molecule useful for cellular adhesion and growth is a drugand it is bonded to the at least one alginate chain section by abiodegradeable bond.
 12. An aqueous composition containing an alginatematerial of claim 1 and water.
 13. An injectable solution for formingcell transplantation matrices comprising a modified alginate, whichcomprises at least one alginate chain section to which is bonded bycovalent bonding at least one molecule useful for cell adhesion andgrowth, and viable cells for said transplantation.
 14. A transplantationmatrix comprising a hydrogel of a modified alginate, which comprises atleast one alginate chain section to which is bonded by covalent bondingat least one molecule useful for cell adhesion and growth, and viablecells for said transplantation.
 15. A method for cell transplantationcomprising administering a mixture of a modified alginate, whichcomprises at least one alginate chain section to which is bonded bycovalent bonding at least one molecule useful for cell adhesion andgrowth, and cells for transplantation.
 16. A polymer comprising, (a) apolymeric backbone section, (b) a side chain comprising polymerizedD-mannuronate monomers, L-guluronate monomers or both D-mannuronate andL-guluronate monomers bonded to said backbone, optionally through alinker, and (c) a biologically active molecule useful for cell adhesionand growth covalently bonded to the side chain.
 17. The polymer of claim16, wherein the biologically active molecule is bonded through a uronicacid residue on the side chain.
 18. The polymer of claim 16, wherein thebiologically active molecule is a cell attachment peptide, a peptidegrowth factor, an enzyme, a proteoglycan or a polysaccharide.
 19. Thepolymer of claim 16, wherein the biologically active molecule is a cellattachment protein.
 20. An injectable solution for forming celltransplantation matrices comprising a polymer according to claim
 19. 21.An injectable solution of claim 20 further comprising viable cells forsaid transplantation.
 22. A cell transplantation matrix comprising ahydrogel of a polymer according to claim 19 and viable cells for saidtransplantation.
 23. The polymer of claim 16, wherein at least one sidechain is bonded through a linker and the linker is a residue of an aminoacid, amino aldehyde, amino alcohol, hydrazine, hydrazide orsemicarbazide.
 24. The polymer of claim 16, wherein the backbone sectionis a poly(vinyl alcohol), poly(ethylene oxide), polypeptide, poly(aminoacid) or poly(uronic acid) polymer section, or modified alginate. 25.The polymer of claim 16, wherein the side chain comprises an oligomericblock unit of D-mannuronate, an oligomeric block unit of L-guluronate,an oligomeric block unit of D-mannuronate and L-guluronate or a mixtureof said block units.
 26. The polymer of claim 16, having a backbonesection with a molecular weight of less than about 50,000.
 27. Thepolymer of claim 26, having side chains each with a molecular weightless than about 50,000.
 28. The polymer of claim 16, having a backbonesection with a molecular weight above 100,000.
 29. The polymer of claim16, wherein the linker provides a biodegradable bond between thebackbone section and the side chain.
 30. The polymer of claim 29,wherein the linker is bonded to the polymeric backbone section by anester group, imine, hydrazone or semicarbazone group.
 31. An aqueouscomposition containing a polymer of claim 16 and water.
 32. A polymercomprising, (a) a polymeric backbone section, and (b) an alginate sidechain, with uronic acid units having carboxylic acid groups, comprisingpolymerized D-mannuronate monomers, L-guluronate monomers or bothD-mannuronate and L-guluronate monomers bonded to said backbone,optionally through a linker, wherein the polymer comprises multiple sidechains wherein at least two of said side chains are crosslinked, thecrosslinking being with a polyfunctional crosslinking agent having atleast two nitrogen-containing functional groups which covalently bond tothe carboxylic acid groups in the uronic acid units of the alginate sidechains.
 33. An alginate material comprising alginate chains withcovalently bonded crosslinking between chains, the crosslinking beingwith a polyfunctional crosslinking agent having at least twonitrogen-containing functional groups which covalently bond tocarboxylic acid groups in uronic acid units of the alginate chains. 34.The alginate material of claim 33, wherein the material is crosslinkedto the extent such that it resumes essentially its original size andshape after compression.
 35. The alginate material of claim 33, whereinthe material additionally is gelled by action of a divalent cation. 36.The alginate material of claim 33, wherein the crosslinking agentcontains at least two amine, hydrazide or semicarbazide functionalgroups, or combinations thereof.
 37. The alginate material of claim 36,wherein the crosslinking agent is lysine or an alkyl ester thereof. 38.The alginate material of claim 33, wherein 1 mole % or more based on themoles of carboxylic acid groups on uronic acid units in the alginatechains are crosslinked.
 39. The alginate material of claim 33, wherein1-20 mole % or more based on the moles of carboxylic acid groups onuronic acid units in the alginate chains are crosslinked.
 40. Thealginate material of claim 33, wherein 5-75 mole % or more based on themoles of carboxylic acid groups on uronic acid units in the alginatechains are crosslinked.
 41. The alginate material of claim 33, whereinthe material is in a viscous liquid form or swellable gel form.
 42. Thealginate material of claim 33, wherein the material is in anon-swellable, compression resistant form having shape memoryproperties.
 43. The alginate material of claim 33, wherein the materialfurther contains a molecule exhibiting cellular interaction activitybonded to an alginate chain.
 44. The alginate material of claim 43,wherein the molecule is a cell adhesion molecule.
 45. The alginatematerial of claim 44, wherein the cell adhesion molecule is a cellattachment peptide, a peptide growth factor, an enzyme, a proteoglycanattachment peptide sequence, a proteoglycan or other polysaccharideexhibiting cell adhesion.
 46. The alginate material of claim 43, whereinthe molecule exhibiting cellular interaction activity is bonded bycovalent bonding to the alginate chain.
 47. A matrix for a cell culturesystem or for tissue engineering composed of the alginate material ofclaim
 33. 48. A method for tissue engineering which comprisesintroducing as a matrix for the tissue an alginate material of claim 33in matrix form.
 49. The method of claim 48, wherein the alginatematerial in matrix form is provided before introduction in a suitablesize and shape, is altered in size or shape during introducing andessentially resumes its suitable size and shape after introducing.
 50. Amethod for cell transplantation comprising administering a combinationof the alginate material of claim 33 and cells for transplantation. 51.A method for treating a human or animal which comprises administeringthereto a modified alginate of claim 33, as a matrix for bone or softtissue replacement.
 52. A polymer comprising, (a) a polymeric backbonesection of a poly(vinyl alcohol), poly(ethylene oxide), polypeptide,poly(amino acid) or poly(uronic acid) polymer, (b) alginate side chains,with uronic acid units having carboxylic acid groups, comprisingpolymerized D-mannuronate monomers, L-guluronate monomers or bothD-mannuronate and L-guluronate monomers bonded to said backbone,optionally through a linker, and (c) at least one biologically activemolecule bonded through a uronic acid unit on an alginate side chain.53. The polymer of claim 52, wherein the biologically active molecule isa cell attachment protein, a cell attachment peptide, a peptide growthfactor, an enzyme, a proteoglycan or a polysaccharide.